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Ben C Creelan,1 Dmitry I Gabrilovich,2 Jhanelle E Gray,1 Charles C Williams,1 Tawee Tanvetyanon,1 Eric B Haura,1 Jeffrey S Weber,3 Geoffrey T Gibney,4 Joseph Markowitz,5 Joel W Proksch,6 Scott A Reisman,6 Mark D McKee,7 Melanie P Chin,6 Colin J Meyer,6 Scott J Antonia11Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Magnolia Drive, Tampa, FL, USA; 2The Wistar Institute, Philadelphia, PA, USA; 3Laura and Isaac Perlmutter Cancer Center, New York, NY, USA; 4Department of Medicine, Georgetown-Lombardi Comprehensive Cancer Center, Washington, DC, USA; 5Department of Cutaneous Oncology, H. Lee Moffitt Cancer Center and Research Institute, Magnolia Drive, Tampa, FL, USA; 6Reata Pharmaceuticals, Inc., Irving, TX, USA; 7AbbVie, Inc., North Chicago, IL, USABackground: Omaveloxolone is a semisynthetic oleanane triterpenoid that potently activates Nrf2 with subsequent antioxidant function. We conducted a first-in-human Phase I clinical trial (NCT02029729) with the primary objectives to determine the appropriate dose for Phase II studies, characterize pharmacokinetic and pharmacodynamic parameters, and assess antitumor activity.Methods: Omaveloxolone was administered orally once daily continuously in a 28-day cycle for patients with stage 4 relapsed/refractory melanoma or non-small cell lung cancer. An accelerated titration design was employed until a grade 2-related adverse event (AE) occurred. A standard 3+3 dose escalation was employed. Single-dose and steady-state plasma pharmacokinetics of the drug were characterized. Downstream Nrf2 activation was assessed in peripheral blood mononuclear cells by quantification of target gene mRNA expression.Results: Omaveloxolone was tested at four dose levels up to 15 mg given orally once daily. No dose-limiting toxicities were detected, and the maximum tolerated dose was not determined. All drug-related AEs were either grade 1 or 2 in severity, and none required clinical action. The most common drug-related AEs were elevated alkaline phosphatase (18%) and anemia (18%). No drug interruptions or reductions were required. Omaveloxolone was rapidly absorbed and exhibited proportional increases in exposure across dose levels. With some exceptions, an overall trend toward time-dependent and dose-dependent activation of Nrf2 antioxidant genes was observed. No confirmed radiologic responses were seen, although one lung cancer subject did have stable disease exceeding 1 year.Conclusions: Omaveloxolone has favorable tolerability at biologically active doses, although this trial had a small sample size which limits definitive conclusions. These findings support further investigation of omaveloxolone in cancer.Keywords: antioxidant inflammation modulator, nitrotyrosine, nitric oxide synthase, melanoma, non-small cell lung cancer, immuno-oncology, myeloid-derived suppressor cells, bardoxolone methyl

Background: The malignancy-risk gene signature is composed of numerous proliferative genes and has been applied to predict breast cancer risk. We hypothesized that the malignancy-risk gene signature has prognostic and predictive value for early-stage non-small cell lung cancer (NSCLC) patients.Methods: The ability of the malignancy-risk gene signature to predict overall survival (OS) of early-stage NSCLC patients was tested using a large NSCLC microarray dataset from the Director's Challenge Consortium (n = 442) and two independent NSCLC microarray datasets (n = 117 and 133, for the GSE13213 and GSE14814 datasets, respectively). An overall malignancy-risk score was generated by principal component analysis to determine the prognostic and predictive value of the signature. An interaction model was used to investigate a statistically significant interaction between adjuvant chemotherapy (ACT) and the gene signature. All statistical tests were two-sided.Results: The malignancy-risk gene signature was statistically significantly associated with OS (P < .001) of NSCLC patients. Validation with the two independent datasets demonstrated that the malignancy-risk score had prognostic and predictive values: Of patients who did not receive ACT, those with a low malignancy-risk score had increased OS compared with a high malignancy-risk score (P = .007 and .01 for the GSE13212 and GSE14814 datasets, respectively), indicating a prognostic value; and in the GSE14814 dataset, patients receiving ACT survived longer in the high malignancy-risk score group (P = .03), and a statistically significant interaction between ACT and the signature was observed (P = .02).Conclusions: The malignancy-risk gene signature was associated with OS and was a prognostic and predictive indicator. The malignancy-risk gene signature could be useful to improve prediction of OS and to identify those NSCLC patients who will benefit from ACT. [ABSTRACT FROM AUTHOR]

Purpose: This phase I study was carried out to determine the phase II recommended dose of tasisulam sodium (hereafter, tasisulam), a novel anticancer agent with a unique mechanism of action.Methods: Tasisulam was administered intravenously, every 21 days, in patients with refractory solid tumors using a three-plus-three dose-escalation schema.Results: Fifty-three patients were enrolled; the first 34 were treated with a flat dose of tasisulam of up to 2,400 mg, the dose level at which all three patients had dose-limiting toxicity (DLT). Controlling for C(max) proved important to reduce the risk of toxicity; therefore, we initially focused on identifying which parameters explained C(max) (end-of-infusion concentration) variability. Pharmacokinetic analysis indicated that C(max) negatively correlates with lean body weight (LBW). Thus, the dosing regimen was revised using a LBW-based algorithm targeting a specific C(max). A loading/chronic dose paradigm was then implemented as pharmacokinetic results revealed a long terminal half-life of tasisulam, likely because of its high-affinity albumin binding. C(max)-based dose escalation was stopped at the 420-μg/mL cohort, in which one of the 16 patients had DLT (transient hepatic transaminase elevation); grade 3/4 hematologic toxicity was noted in later cycles in three patients. Although response was not a primary objective, 33% of heavily pretreated patients with post-dose radiological assessments had stable disease.Conclusion: Implementation of a novel targeted C(max)-based dosing regimen allowed for the recommendation of a phase II tasisulam dose (loading dose of 420 μg/mL targeted C(max) with all subsequent doses administered at 65% of chronic dose given every 21 days) despite pharmacological challenges posed by high albumin binding. [ABSTRACT FROM AUTHOR]

Src family kinases (SFKs) have a critical role in cell adhesion, invasion, proliferation, survival, and angiogenesis during tumor development. SFKs comprise nine family members that share similar structure and function. Overexpression or high activation of SFKs occurs frequently in tumor tissues and they are central mediators in multiple signaling pathways that are important in oncogenesis. SFKs can interact with tyrosine kinase receptors, such as EGFR and the VEGF receptor. SFKs can affect cell proliferation via the Ras/ERK/MAPK pathway and can regulate gene expression via transcription factors such as STAT molecules. SFKs can also affect cell adhesion and migration via interaction with integrins, actins, GTPase-activating proteins, scaffold proteins, such as p130(CAS) and paxillin, and kinases such as focal adhesion kinases. Furthermore, SFKs can regulate angiogenesis via gene expression of angiogenic growth factors, such as fibroblast growth factor, VEGF, and interleukin 8. On the basis of these important findings, small-molecule SFK inhibitors have been developed and are undergoing early phase clinical testing. In preclinical studies these agents can suppress tumor growth and metastases. The agents seem to be safe in humans and could add to the therapeutic arsenal against subsets of cancers. [ABSTRACT FROM AUTHOR]

Purpose: Overexpression of Bcl-2 family members as well as deregulated apoptosis pathways are known hallmarks of lung cancer. Non-small cell lung cancer (NSCLC) cells are typically resistant to cytotoxic chemotherapy and approaches that alter the balance between pro-survival and pro-death Bcl-2 family members have shown promise in preclinical models of NSCLC.Methods: Here we evaluated the effects of a novel pan-Bcl-2 inhibitor GX15-070 on NSCLC survival and when combined with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors as well as traditional cytotoxic agents. GX15-070 is a small molecule agent that binds anti-apoptotic Bcl-2 proteins and interferes with their ability to interact with pro-apoptotic proteins. We evaluated the effect of GX15-070 and correlated the effect on EGFR status as well as Bcl-2 family protein expression.Results: We show that GX15-070 can disrupt Mcl-1:Bak interactions in lung cancer cells. We identified differential sensitivity of a panel of lung cancer cells to GX15-070 and no clear relationship existed between EGFR status or Bcl-2 family protein expression and sensitivity to GX15-070. GX15-070 could induce apoptosis in a subset of lung cancer cell lines and this correlated with the effects on cell viability. GX15-070 combined with gefitinib was synergistic in a cell line dependent on EGFR for survival but GX15-070 could not reverse resistance to gefitinib in cell lines not dependent on EGFR for survival. Finally, we observed synergy between GX15-070 and cisplatin in NSCLC cells.Conclusions: Based on these results, GX15-070 can trigger apoptosis in NSCLC cells and can enhance chemotherapy-induced death. These data suggest that clinical trials with GX15-070 in combination with cytotoxic chemotherapy are indicated. [ABSTRACT FROM AUTHOR]

Cancer-associated fibroblasts (CAFs) are associated with tumor progression and modulate drug sensitivity of cancer cells. However, the underlying mechanisms are often incompletely understood and crosstalk between tumor cells and CAFs involves soluble secreted as well as adhesion proteins. Interrogating a panel of non-small cell lung cancer (NSCLC) cell lines driven by EML4-ALK fusions, we observed substantial CAF-mediated drug resistance to clinical ALK tyrosine kinase inhibitors (TKIs). Array-based cytokine profiling of fibroblast-derived conditioned- media identified HGF-MET signaling as a major contributor to CAF-mediated paracrine resistance that can be overcome by MET TKIs. However, 'Cell Type specific labeling using Amino acid Precursors' (CTAP)-based expression and phosphoproteomics in direct coculture also highlighted a critical role for the fibronectin-integrin pathway. Flow cytometry analysis confirmed activation of integrin β1 (ITGB1) in lung cancer cells by CAF coculture. Treatment with pharmacological inhibitors, cancer cell-specific silencing or CRISPR-Cas9-mediated knockout of ITGB1 overcame adhesion protein-mediated resistance. Concurrent targeting of MET and integrin signaling effectively abrogated CAF-mediated resistance of EML4-ALK -driven NSCLC cells to ALK TKIs in vitro . Consistently, combination of the ALK TKI alectinib with the MET TKI capmatinib and/or the integrin inhibitor cilengitide was significantly more efficacious than single agent treatment in suppressing tumor growth using an in vivo EML4-ALK -dependent allograft mouse model of NSCLC. In summary, these findings emphasize the complexity of resistance-associated crosstalk between CAFs and cancer cells, which can involve multiple concurrent signaling pathways, and illustrate how comprehensive elucidation of paracrine and juxtacrine resistance mechanisms can inform on more effective therapeutic approaches.

Immunotherapy response is associated with the presence of conventional dendritic cells (cDCs). cDC type 1 (cDC1) is critically important for CD8+ T cell activation, cDC type 2 (cDC2) regulates CD4+ T cell responses, and mature regulatory cDCs may dampen T cell responses in the tumor microenvironment (TME). However, we lack a clear understanding of cDC distribution in the human TME, cDC prevalence in metastatic sites, and cDC differences in early- versus late-stage disease. Rapid autopsy specimens of 10 patients with lung adenocarcinoma were evaluated to detect cDCs and immune cells via multiplex immunofluorescence using 18 markers and 42 tumors. First, we found that T cells, cDC1, and cDC2 were confined to stroma, whereas mature regulatory DCs were enriched in tumor, suggesting unique localization-specific functions. Second, lung and lymph node tumors were more enriched in T cells and cDCs than liver tumors, underscoring differences in the TME of metastatic sites. Third, although the proportion of T cells and cDC1 did not differ in different stages, an increase in the proportion of cDC2 and macrophages in late stage suggests potential differences in regulation of T cell responses in different stages. Collectively, these findings provide new, to our knowledge, insights into cDC biology in human cancer that may have important therapeutic implications., (Copyright © 2024 by The American Association of Immunologists, Inc.)

This study describes the validation of a clinical RNA expression panel with evaluation of concordance between gene copy gain by a next-generation sequencing (NGS) assay and high gene expression by an RNA expression panel. The RNA Salah Targeted Expression Panel (RNA STEP) was designed with input from oncologists to include 204 genes with utility for clinical trial prescreening and therapy selection. RNA STEP was validated with the nanoString platform using remnant formalin-fixed, paraffin-embedded-derived RNA from 102 patients previously tested with a validated clinical NGS panel. The repeatability, reproducibility, and concordance of RNA STEP results with NGS results were evaluated. RNA STEP demonstrated high repeatability and reproducibility, with excellent correlation (r > 0.97, P < 0.0001) for all comparisons. Comparison of RNA STEP high gene expression (log2 ratio ≥ 2) versus NGS DNA-based gene copy number gain (copies ≥ 5) for 38 mutually covered genes revealed an accuracy of 93.0% with a positive percentage agreement of 69.4% and negative percentage agreement of 93.8%. Moderate correlation was observed between platforms (r = 0.53, P < 0.0001). Concordance between high gene expression and gene copy number gain varied by specific gene, and some genes had higher accuracy between assays. Clinical implementation of RNA STEP provides gene expression data complementary to NGS and offers a tool for prescreening patients for clinical trials., Competing Interests: Disclosure Statement T.A.B. and J.M.K. have a contract with Bristol Myers Squibb unrelated to this research. E.B.H. consults for Revolution Medicines, Janssen, Ellipses Pharma, Amgen, Kanaph Therapeutics, and ORI Capital, and holds a patent for protein-protein interactions as biomarkers; and research funding from Janssen, Novartis, Revolution Medicines, AstraZeneca, Spectrum Pharmaceuticals, and Genentech, all unrelated to this research. A.N.S. has clinical trial funding to his institution from AstraZeneca, BioAtla, Daiichi Sankyo, Eli Lily, Genentech, Genmab, Memgen, Mersana, Novartis, and Turning Point Therapeutics; and has participated in advisory boards for Daiichi Sankyo, Eli Lilly, and Zymeworks, all unrelated to this research., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocytosis, and primary myelofibrosis, are clonal hematopoietic neoplasms driven by mutationally activated signaling by the JAK2 tyrosine kinase. Although JAK2 inhibitors can improve MPN patients' quality of life, they do not induce complete remission as disease-driving cells persistently survive therapy. ERK activation has been highlighted as contributing to JAK2 inhibitor persistent cell survival. As ERK is a component of signaling by activated RAS proteins and by JAK2 activation, we sought to inhibit RAS activation to enhance responses to JAK2 inhibition in preclinical MPN models. We found the SHP2 inhibitor RMC-4550 significantly enhanced growth inhibition of MPN cell lines in combination with the JAK2 inhibitor ruxolitinib, effectively preventing ruxolitinib persistent growth, and the growth and viability of established ruxolitinib persistent cells remained sensitive to SHP2 inhibition. Both SHP2 and JAK2 inhibition diminished cellular RAS-GTP levels, and their concomitant inhibition enhanced ERK inactivation and increased apoptosis. Inhibition of SHP2 inhibited the neoplastic growth of MPN patient hematopoietic progenitor cells and exhibited synergy with ruxolitinib. RMC-4550 antagonized MPN phenotypes and increased survival of an MPN mouse model driven by MPL-W515L. The combination of RMC-4550 and ruxolitinib, which was safe and tolerated in healthy mice, further inhibited disease compared to ruxolitinib monotherapy, including extending survival. Given SHP2 inhibitors are undergoing clinical evaluation in patients with solid tumors, our preclinical findings suggest that SHP2 is a candidate therapeutic target with potential for rapid translation to clinical assessment to improve current targeted therapies for MPN patients., (© 2024 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Multiple tyrosine kinase inhibitors (TKIs) are often developed for the same indication. However, their relative overall efficacy is frequently incompletely understood and they may harbor unrecognized targets that cooperate with the intended target. We compared several ROS1 TKIs for inhibition of ROS1-fusion-positive lung cancer cell viability, ROS1 autophosphorylation and kinase activity, which indicated disproportionately higher cellular potency of one TKI, lorlatinib. Quantitative chemical and phosphoproteomics across four ROS1 TKIs and differential network analysis revealed that lorlatinib uniquely impacted focal adhesion signaling. Functional validation using pharmacological probes, RNA interference, and CRISPR-Cas9 knockout uncovered a polypharmacology mechanism of lorlatinib by dual targeting ROS1 and PYK2, which form a multiprotein complex with SRC. Rational multi-targeting of this complex by combining lorlatinib with SRC inhibitors exhibited pronounced synergy. Taken together, we show that systems pharmacology-based differential network analysis can dissect mixed canonical/non-canonical polypharmacology mechanisms across multiple TKIs enabling the design of rational drug combinations., Competing Interests: Declaration of interests R.C.D. is an employee and shareholder of Rain Oncology and reports licensing fees from Voronoi, Takeda, Loxo@Lilly, Foundation Medicine, Histocyte, Black Diamond, and ThermoFisher. JMK reports support from Bristol-Myers Squibb on an unrelated project. E.B.H. serves in a consulting or advisory role to Amgen, Ellipses Pharma, Janssen Oncology, Janssen Research & Development and Revolution Medicines; reports research funding (paid to his institution) from AstraZeneca, Genentech, Incyte, Janssen, Novartis, Revolution Medicines and Spectrum Pharmaceuticals; and reports patents, royalties or other intellectual property from ProteinProtein Interactions as Biomarkers Patent., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Despite the initial benefit from tyrosine kinase inhibitors (TKI) targeting oncogenic ALK and ROS1 gene fusions in non-small cell lung cancer, complete responses are rare and resistance ultimately emerges from residual tumor cells. Although several acquired resistance mechanisms have been reported at the time of disease progression, adaptative resistance mechanisms that contribute to residual diseases before the outgrowth of tumor cells with acquired resistance are less clear. For the patients who have progressed after TKI treatments, but do not demonstrate ALK/ROS1 kinase mutations, there is a lack of biomarkers to guide effective treatments. Herein, we found that phosphorylation of MIG6, encoded by the ERRFI1 gene, was downregulated by ALK/ROS1 inhibitors as were mRNA levels, thus potentiating EGFR activity to support cell survival as an adaptive resistance mechanism. MIG6 downregulation was sustained following chronic exposure to ALK/ROS1 inhibitors to support the establishment of acquired resistance. A higher ratio of EGFR to MIG6 expression was found in ALK TKI-treated and ALK TKI-resistant tumors and correlated with the poor responsiveness to ALK/ROS1 inhibition in patient-derived cell lines. Furthermore, we identified and validated a MIG6 EGFR-binding domain truncation mutation in mediating resistance to ROS1 inhibitors but sensitivity to EGFR inhibitors. A MIG6 deletion was also found in a patient after progressing to ROS1 inhibition. Collectively, this study identifies MIG6 as a novel regulator for EGFR-mediated adaptive and acquired resistance to ALK/ROS1 inhibitors and suggests EGFR to MIG6 ratios and MIG6-damaging alterations as biomarkers to predict responsiveness to ALK/ROS1 and EGFR inhibitors., (©2023 The Authors; Published by the American Association for Cancer Research.)

Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The optimal treatment sequencing for patients with metastatic epidermal growth factor receptor ( EGFR )-mutant non-small cell lung cancer (NSCLC) remains a subject of debate. In the United States, osimertinib is the preferred EGFR tyrosine kinase inhibitor (TKI) in the first-line setting. However, small retrospective studies suggest that alternative EGFR TKI sequencing strategies may produce similar outcomes. This study aimed to compare the outcomes of patients with metastatic NSCLC harboring an EGFR exon 19 deletion or exon 21 L858R mutation treated with osimertinib vs. afatinib as first-line therapy., Methods: This retrospective, single-institution study examined 86 patients with metastatic EGFR -mutant NSCLC treated with either afatinib (n=15) or osimertinib (n=71) in the first-line setting. The primary outcome was progression-free survival (PFS), and secondary endpoints included time on EGFR TKI, overall survival (OS), and the incidence of adverse events (AEs)., Results: There was no difference in the PFS (median: 27.9 vs. 29.0 months, P=0.75), OS (P=0.18), and the median time on first-line EGFR TKI (23.9 vs. 15.2 months, P=0.10) between the afatinib and osimertinib groups, respectively. The number of AEs was also similar between the two treatment groups (P=0.17)., Conclusions: In this real-world retrospective study, there were no differences in PFS or OS between patients treated with afatinib or osimertinib in the first-line setting. These findings should be further investigated in larger prospective studies., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-686/coif). B.P. has received research support to the institution from Bristol Myers Squibb, speaker honoraria from BioAscend, Merck, MJH Life Science, Play to Know AG, Grupo Pardini, GBOT, Doctaforum, Foundation Medicine, and has done consulting/advisory board work with Guidepoint, Guardant Health, Foundation Medicine, Illumina, BMS, Regeneron, AstraZeneca, and AH Merus. B.P. reports funding to the institution from the Bristol Myers Squibb Foundation/the Robert A. Winn Diversity in Clinical Trials Awards Program. D.M. has done consulting/advisory board work with Dedham Group, Precisionxtract, Pfizer, Eli Lily, and Takeda, and has received speaker honorarium from Jazz Pharmaceuticals. M.S. has done consulting/advisory board work with Jazz Pharmaceuticals and GlaxoSmithKline. M.S. reports funding from Daiichi Sankyo/Lilly and funding to the institution from Merck Serono, Vaccinex, Nektar, Amphivena, and Pfizer. A.S. served as speaker honoraria for MJH Life Science and HMP Global and has done consulting/advisory board work with Daiichi Sankyo, Eli Lilly, and Zymeworks. A.S. reports funding to the institution from Genetech, Genmab, Turning Point Therapeutics, Novartis, Eli Lily, Memgen, Mersana, BioAlta, AstraZeneca, Daiichi Sankyo. B.C. has received speaking fees from AstraZeneca, ARIAD Pharmaceuticals and Hoffmann-La Roche and consultant fees from Xilio, Achilles, ER Squibb, Hoffmann-La Roche, AstraZeneca, AbbVie, KSQ Therapeutics, GlaxoSmithKline, Gilead Sciences, Celgene. B.C. has received speaking fees from AstraZeneca plc, ARIAD Pharmaceuticals, Hoffmann-La Roche Ag, and OMNI Health Media LLC, and consultant fees from AbbVie, Achilles Therapeutics plc, AstraZeneca plc, Celgene, ER Squibb, G1 Therapeutics Inc, Gilead Sciences, GlaxoSmithKline, Hoffmann-La Roche, KSQ Therapeutics, MJH Life Sciences, Regeneron Pharmaceuticals Inc., Techspert IO Ltd., and Xilio and has received research funding from ER Squibb LLC and Iovance Biotherapeutics Inc., and has a patent applications filed US201962865697P and US202062976867P outside the submitted work. A.C. has received speaker bureau funding from Genentech, Merck, Takeda, Novartis, Boehringer Ingelheim, and Celgene and research funding from Novartis and Bristol-Myers Squibb. E.B.H. received speaker honoraria for ASCO 2022 Annual Meeting and the 35th Aspen Cancer Conference, received consulting fees from Ellipses, Kanaph Therapeutics, Inc., and ORI Capital II, Inc. E.B.H. has done data safety monitoring/advisory board work with Amgen, Jassen, RevMed, and reports grants to the institution from Revolution Medicine. J.E.G. has received research support AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Genetech, GI Therapeutics, Ludwig Institute of Cancer Research, Merck, Novartis, Pfizer. J.E.G. has done consulting/advisory board work with AbbVie, AstraZeneca, Axiom HC Strategies, Blueprint Medicines, Bristol Myers Squibb, Celgene Corp, Daiichi Sankyo, EMD Serono-Merck KGaA, Genentech, Inivata, Janssen Scientific Affairs, Jazz Pharmaceuticals, Loxo Oncology Inc, Merck, Novartis, OncoCyte Biotechnology Company, Sanofi Pharmaceuticals, Takeda Pharmaceuticals, Triptych Health Partners. G.S. reports funding from AstraZeneca, NovoCure, and Syneos Health, consulting fees and speaker honoraria from AstraZeneca, NovoCure, OncLive, and DAVA Oncology. G.S. has done data safety monitoring/advisory board work with AstraZeneca, NovoCure, and FLASCO. The other authors have no conflicts of interest to declare., (2023 Journal of Thoracic Disease. All rights reserved.)

Patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) often develop resistance to current standard third-generation EGFR tyrosine kinase inhibitors (TKIs); no targeted treatments are approved in the osimertinib-relapsed setting. In this open-label, dose-escalation and dose-expansion phase 1 trial, the potential for improved anti-tumor activity by combining amivantamab, an EGFR-MET bispecific antibody, with lazertinib, a third-generation EGFR TKI, was evaluated in patients with EGFR-mutant NSCLC whose disease progressed on third-generation TKI monotherapy but were chemotherapy naive (CHRYSALIS cohort E). In the dose-escalation phase, the recommended phase 2 combination dose was established; in the dose-expansion phase, the primary endpoints were safety and overall response rate, and key secondary endpoints included progression-free survival and overall survival. The safety profile of amivantamab and lazertinib was generally consistent with previous experience of each agent alone, with 4% experiencing grade ≥3 events; no new safety signals were identified. In an exploratory cohort of 45 patients who were enrolled without biomarker selection, the primary endpoint of investigator-assessed overall response rate was 36% (95% confidence interval, 22-51). The median duration of response was 9.6 months, and the median progression-free survival was 4.9 months. Next-generation sequencing and immunohistochemistry analyses identified high EGFR and/or MET expression as potential predictive biomarkers of response, which will need to be validated with prospective assessment. ClinicalTrials.gov identifier: NCT02609776 ., (© 2023. The Author(s).)

Background: Activating mutations in EGFR or KRAS are highly prevalent in NSCLC, share activation of the MAPK pathway and may be amenable to combination therapy to prevent negative feedback activation., Methods: In this phase 1/1B trial, we tested the combination of binimetinib and erlotinib in patients with advanced NSCLC with at least 1 prior line of treatment (unless with activating EGFR mutation which could be treatment-naïve). A subsequent phase 1B expansion accrued patients with either EGFR- or KRAS-mutation using the recommended phase 2 dose (RP2D) from Phase 1. The primary objective was to evaluate the safety of binimetinib plus erlotinib and establish the RP2D., Results: 43 patients enrolled (dose-escalation = 23; expansion = 20). 17 harbored EGFR mutation and 22 had KRAS mutation. The RP2D was erlotinib 100 mg daily and binimetinib 15 mg BID × 5 days/week. Common AEs across all doses included diarrhea (69.8%), rash (44.2%), fatigue (32.6%), and nausea (32.6%), and were primarily grade 1/2. Among KRAS mutant patients, 1 (5%) had confirmed partial response and 8 (36%) achieved stable disease as best overall response. Among EGFR mutant patients, 9 were TKI-naïve with 8 (89%) having partial response, and 8 were TKI-pretreated with no partial responses and 1 (13%) stable disease as best overall response., Conclusions: Binimetinib plus erlotinib demonstrated a manageable safety profile and modest efficacy including one confirmed objective response in a KRAS mutant patient. While clinical utility of this specific combination was limited, these results support development of combinations using novel small molecule inhibitors of RAS, selective EGFR- and other MAPK pathway inhibitors, many of which have improved therapeutic indices., Clinical Trial Registration: NCT01859026., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ANS declares serving on advisory board for Eli Lilly, Daiichi Sankyo and Zymeworks, and research funding from Eli Lilly, Novartis, Daiichi Sankyo, Genentech, AstraZeneca, Memgen, Mersana, Turning Point Therapeutics, BioAtla and Genmab to his institution, BCC declares research support from Clinigen Group and Bristol-Myers Squibb, speakers’ bureau from Novartis and AstraZeneca, travel reimbursement from AstraZeneca, and honoraria from Achilles Therapeutics, Roche, AstraZeneca, Instil Bio, Bristol-Myers Squibb, AAC declares research support from Bristol Myers Squibb, AstraZeneca, and Novartis, and speaker, consultant and/or advisor honoraria from Blueprint, Takeda, Genentech, Pfizer, Merck, Astra Zeneca, Janssen and Jazz, SJA declares consulting from Memgen, Achilles Therapeutics, Amgen, Celsius Therapeutics, G1 Therapeutics, GlaxoSmithKline, Glympse Bio, RAPT Therapeutics, Samyang, Venn Therapeutics, Tarus Therapeutics, Bristol-Myers Squibb, EMD Serono, and Merck, patent for Oncolytic Virus, and travel expenses from RAPT Therapeutics, Celsius Therapeutics, Amgen and Achilles Therapeutics, MRC declares consulting fees from Jazz Pharmaceuticals, OncLive, Evicore, Targeted Oncology, and Journal of Clinical Pathways, and research funding from Jazz Pharmaceuticals, Daiichi Sankyo, and Pfizer to his institution, EBH declares consulting from Revolution Medicines, Janssen, Ellipses Pharma, Amgen, Kanaph Therapeutics, and ORI Capital, patent for Protein-Protein Interactions as Biomarkers, and research funding from Janssen, Novartis, Revolution Medicines, AstraZeneca, Spectrum Pharmaceuticals, and Genentech, JEG declares consulting fees and/or honoraria from AbbVie, AstraZeneca, Axiom HC Strategies, Jazz, Loxo Oncology, Blueprint Medicines, Bristol-Myers Squibb, Merck, Novartis, OncoCyte Biotech, Celgene Daiichi Sankyo, EMD Serono, Sanofi, Takeda, Genentech, Inivata, Janssen and Triptych Health Partners, and research support from AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Novartis, Pfizer, Genentech, G1 Therapeutics, Ludwig Institute of Cancer Research, and Merck. The other authors declare no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: ERBB2 or HER2 alterations are found in approximately 2% to 5% of NSCLCs; most are exon 20 insertion mutations. The efficacy and safety of poziotinib, an oral tyrosine kinase inhibitor, were assessed in patients with treatment-naive NSCLC whose tumors harbor HER2 exon 20 insertions., Methods: ZENITH20 is an open-label, multicohort, multicenter, global, phase 2 trial. ZENITH20-C4 enrolled treatment-naive patients with NSCLC with tumors harboring HER2 exon 20 insertions. Poziotinib was administered 16 mg once daily (QD) or 8 mg twice daily (BID). The primary end point was objective response rate (ORR) by independent central review. Secondary and exploratory end points included disease control rate, duration of response, progression-free survival, and safety., Results: A total of 80 patients (16 mg QD, n = 47; 8 mg BID, n = 33) were treated in ZENITH20-C4. ORR was 39% (95% confidence interval [CI]: 28%-50%; 31 of 80), with a disease control rate of 73% (95% CI: 61%-82%; 58 of 80); 80% of the patients experienced tumor reduction. Median duration of response was 5.7 (95% CI: 4.6-11.9) months, and median progression-free survival was 5.6 (95% CI: 5.4-7.3) months. The most common grade 3 treatment-related adverse events were rash (QD, 45%; BID, 39%), stomatitis (QD, 21%; BID, 15%), and diarrhea (QD, 15%; BID, 21%). Among all subtypes of HER2 exon 20 insertions, seven patients (9%) harboring tumors with G778&#95;P780dupGSP had the best clinical outcomes (ORR, 71%)., Conclusions: Poziotinib was found to have clinically meaningful efficacy with a manageable toxicity profile for patients with treatment-naive NSCLC harboring HER2 exon 20 mutations., (Copyright © 2023. Published by Elsevier Inc.)

Purpose: We hypothesized that concurrent ipilimumab with chemoradiationtherapy (chemoRT) followed by maintenance nivolumab would be safe for patients with unresectable stage III non-small cell lung cancer (NSCLC). We aimed to assess the safety (phase 1) and the 12-month progression-free survival (PFS) (phase 2) in a multi-institution prospective trial., Methods and Materials: Eligible patients had unresectable stage III NSCLC. The treatment included platinum doublet chemotherapy with concurrent thoracic radiation therapy to 60 Gy in 30 fractions and ipilimumab (1 mg/kg) delivered during weeks 1 and 4. After chemoRT, maintenance nivolumab (480 mg) was given every 4 weeks for up to 12 cycles. Adverse events (AEs) were assessed according to the Common Terminology Criteria for Adverse Events, version 5.0. Survival analyses were performed with Kaplan Meier (KM) methods and log-rank tests., Results: The trial was discontinued early after enrolling 19 patients without proceeding to the phase 2 component because of unacceptable toxicity. Sixteen patients (84%) had grade ≥3 (G3+) possible treatment-related toxicity, most commonly pulmonary AEs (n = 8, 42%). Fourteen patients (74%) discontinued study therapy early because of AEs (n = 12, 63%) or patient choice (n = 2, 11%). Eleven patients (58%) experienced G2+ pulmonary toxicity with median time to onset 4.1 months (95% CI 2.6-not reached [NR]), and 12-month freedom from G2+ pulmonary toxicity 37% (95% CI, 16-59). Five patients had G5 AEs, including 3 with G5 pulmonary AEs (1 respiratory failure with pneumonitis and pulmonary embolism, 1 pneumonia/chronic obstructive pulmonary disease exacerbation, 1 pulmonary fibrosis). Despite toxicities, the median PFS was 19.2 months (95% CI 6.1-NR) and the median overall survival was NR (95% CI 6.1-NR) with median follow-up of 30.1 months by the reverse KM method., Conclusions: Concurrent ipilimumab with chemoRT for unresectable stage III NSCLC is associated with pulmonary toxicity that may limit opportunities for improved outcomes. Future studies aiming to incorporate ipilimumab or other anti-CTLA4 therapies into management of unresectable stage III NSCLC should consider careful measures to minimize toxicity risk., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has widespread clinical use for detection of inborn errors of metabolism, therapeutic drug monitoring, and numerous other applications. This technique detects proteolytic peptides as surrogates for protein biomarker expression, mutation, and post-translational modification in individual clinical assays and in cancer research with highly multiplexed quantitation across biological pathways. LC-MRM for protein biomarkers must be translated from multiplexed research-grade panels to clinical use. LC-MRM panels provide the capability to quantify clinical biomarkers and emerging protein markers to establish the context of tumor phenotypes that provide highly relevant supporting information. An application to visualize and communicate targeted proteomics data will empower translational researchers to move protein biomarker panels from discovery to clinical use. Therefore, we have developed a web-based tool for targeted proteomics that provides pathway-level evaluations of key biological drivers ( e.g. , EGFR signaling), signature scores (representing phenotypes) ( e.g., EMT), and the ability to quantify specific drug targets across a sample cohort. This tool represents a framework for integrating summary information, decision algorithms, and risk scores to support Physician-Interpretable Phenotypic Evaluation in R (PIPER) that can be reused or repurposed by other labs to communicate and interpret their own biomarker panels.

Metastasis poses a major challenge in cancer management, including EML4-ALK-rearranged non-small cell lung cancer (NSCLC). As cell migration is a critical step during metastasis, we assessed the anti-migratory activities of several clinical ALK inhibitors in NSCLC cells and observed differential anti-migratory capabilities despite similar ALK inhibition, with brigatinib displaying superior anti-migratory effects over other ALK inhibitors. Applying an unbiased in situ mass spectrometry-based chemoproteomics approach, we determined the proteome-wide target profile of brigatinib in EML4-ALK+ NSCLC cells. Dose-dependent and cross-competitive chemoproteomics suggested MARK2 and MARK3 as relevant brigatinib kinase targets. Functional validation showed that combined pharmacological inhibition or genetic modulation of MARK2/3 inhibited cell migration. Consistently, brigatinib treatment induced inhibitory YAP1 phosphorylation downstream of MARK2/3. Collectively, our data suggest that brigatinib exhibits unusual cross-phenotype polypharmacology as, despite similar efficacy for inhibiting EML4-ALK-dependent cell proliferation as other ALK inhibitors, it more effectively prevented migration of NSCLC cells due to co-targeting of MARK2/3., (© 2023 Wiley-VCH GmbH.)

Background: Amivantamab, a fully humanized EGFR-MET bispecific antibody, has antitumor activity in diverse EGFR- and MET-driven non-small cell lung cancer (NSCLC) and a safety profile consistent with associated on-target activities. Infusion-related reaction(s) (IRR[s]) are reported commonly with amivantamab. We review IRR and subsequent management in amivantamab-treated patients., Methods: Patients treated with the approved dose of intravenous amivantamab (1050 mg, <80 kg; 1400 mg, ≥80 kg) in CHRYSALIS-an ongoing, phase 1 study in advanced EGFR-mutated NSCLC-were included in this analysis. IRR mitigations included split first dose (350 mg, day 1 [D1]; remainder, D2), reduced initial infusion rates with proactive infusion interruption, and steroid premedication before initial dose. For all doses, pre-infusion antihistamines and antipyretics were required. Steroids were optional after the initial dose., Results: As of 3/30/2021, 380 patients received amivantamab. IRRs were reported in 256 (67%) patients. Signs/symptoms of IRR included chills, dyspnea, flushing, nausea, chest discomfort, and vomiting. Most of the 279 IRRs were grade 1 or 2; grade 3 and 4 IRR occurred in 7 and 1 patients, respectively. Most (90%) IRRs occurred on cycle 1, D1 (C1D1); median time-to-first-IRR onset during C1D1 was 60 min; and first-infusion IRRs did not compromise subsequent infusions. Per protocol, IRR was mitigated on C1D1 with holding of infusion (56% [214/380]), reinitiating at reduced rate (53% [202/380]), and aborting infusion (14% [53/380]). C1D2 infusions were completed in 85% (45/53) of patients who had C1D1 infusions aborted. Four patients (1% [4/380]) discontinued treatment due to IRR. In studies aimed at elucidating the underlying mechanism(s) of IRR, no pattern was observed between patients with versus without IRR., Conclusion: IRRs with amivantamab were predominantly low grade and limited to first infusion, and rarely occurred with subsequent dosing. Close monitoring for IRR with the initial amivantamab dose and early intervention at first IRR signs/symptoms should be part of routine amivantamab administration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Janssen Research and Development LLC. Published by Elsevier B.V. All rights reserved.)

We analyzed large-scale post-translational modification (PTM) data to outline cell signaling pathways affected by tyrosine kinase inhibitors (TKIs) in ten lung cancer cell lines. Tyrosine phosphorylated, lysine ubiquitinated, and lysine acetylated proteins were concomitantly identified using sequential enrichment of post translational modification (SEPTM) proteomics. Machine learning was used to identify PTM clusters that represent functional modules that respond to TKIs. To model lung cancer signaling at the protein level, PTM clusters were used to create a co-cluster correlation network (CCCN) and select protein-protein interactions (PPIs) from a large network of curated PPIs to create a cluster-filtered network (CFN). Next, we constructed a Pathway Crosstalk Network (PCN) by connecting pathways from NCATS BioPlanet whose member proteins have PTMs that co-cluster. Interrogating the CCCN, CFN, and PCN individually and in combination yields insights into the response of lung cancer cells to TKIs. We highlight examples where cell signaling pathways involving EGFR and ALK exhibit crosstalk with BioPlanet pathways: Transmembrane transport of small molecules; and Glycolysis and gluconeogenesis. These data identify known and previously unappreciated connections between receptor tyrosine kinase (RTK) signal transduction and oncogenic metabolic reprogramming in lung cancer. Comparison to a CFN generated from a previous multi-PTM analysis of lung cancer cell lines reveals a common core of PPIs involving heat shock/chaperone proteins, metabolic enzymes, cytoskeletal components, and RNA-binding proteins. Elucidation of points of crosstalk among signaling pathways employing different PTMs reveals new potential drug targets and candidates for synergistic attack through combination drug therapy., Competing Interests: JK was funded in part by a sponsored research agreement with Bristol Myers Squibb unrelated to this work., (Copyright: © 2023 Ross et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: Multimodality treatment for resectable non-small cell lung cancer has long remained at a therapeutic plateau. Immune checkpoint inhibitors are highly effective in advanced non-small cell lung cancer and promising preoperatively in small clinical trials for resectable non-small cell lung cancer. This large multicenter trial tested the safety and efficacy of neoadjuvant atezolizumab and surgery., Methods: Patients with stage IB to select IIIB resectable non-small cell lung cancer and Eastern Cooperative Oncology Group performance status 0/1 were eligible. Patients received atezolizumab 1200 mg intravenously every 3 weeks for 2 cycles or less followed by resection. The primary end point was major pathological response in patients without EGFR/ALK+ alterations. Pre- and post-treatment computed tomography, positron emission tomography, pulmonary function tests, and biospecimens were obtained. Adverse events were recorded by Common Terminology Criteria for Adverse Events v.4.0., Results: From April 2017 to February 2020, 181 patients were entered in the study. Baseline characteristics were mean age, 65.1 years; female, 93 of 181 (51%); nonsquamous histology, 112 of 181 (62%); and clinical stages IIB to IIIB, 147 of 181 (81%). In patients without EGFR/ALK alterations who underwent surgery, the major pathological response rate was 20% (29/143; 95% confidence interval, 14-28) and the pathological complete response rate was 6% (8/143; 95% confidence interval, 2-11). There were no grade 4/5 treatment-related adverse events preoperatively. Of 159 patients (87.8%) undergoing surgery, 145 (91%) had pathologic complete resection. There were 5 (3%) intraoperative complications, no intraoperative deaths, and 2 postoperative deaths within 90 days, 1 treatment related. Median disease-free and overall survival have not been reached., Conclusions: Neoadjuvant atezolizumab in resectable stage IB to IIIB non-small cell lung cancer was well tolerated, yielded a 20% major pathological response rate, and allowed safe, complete surgical resection. These results strongly support the further development of immune checkpoint inhibitors as preoperative therapy in locally advanced non-small cell lung cancer., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Photoreactive fragment-like probes have been applied to discover target proteins that constitute novel cellular vulnerabilities and to identify viable chemical hits for drug discovery. Through forming covalent bonds, functionalized probes can achieve stronger target engagement and require less effort for on-target mechanism validation. However, the design of probe libraries, which directly affects the biological target space that is interrogated, and effective target prioritization remain critical challenges of such a chemical proteomic platform. In this study, we designed and synthesized a diverse panel of 20 fragment-based probes containing natural product-based privileged structural motifs for small-molecule lead discovery. These probes were fully functionalized with orthogonal diazirine and alkyne moieties and used for protein crosslinking in live lung cancer cells, target enrichment via "click chemistry," and subsequent target identification through label-free quantitative liquid chromatography-tandem mass spectrometry analysis. Pair-wise comparison with a blunted negative control probe and stringent prioritization via individual cross-comparisons against the entire panel identified glutathione S -transferase zeta 1 (GSTZ1) as a specific and unique target candidate. DepMap database query, RNA interference-based gene silencing, and proteome-wide tyrosine reactivity profiling suggested that GSTZ1 cooperated with different oncogenic alterations by supporting survival signaling in refractory non-small cell lung cancer cells. This finding may form the basis for developing novel GSTZ1 inhibitors to improve the therapeutic efficacy of oncogene-directed targeted drugs. In summary, we designed a novel fragment-based probe panel and developed a target prioritization scheme with improved stringency, which allows for the identification of unique target candidates, such as GSTZ1 in refractory lung cancer.

We report a novel method to inhibit epidermal growth factor receptor (EGFR) signaling using custom morpholino antisense oligonucleotides (ASOs) to drive expression of dominant negative mRNA isoforms of EGFR by ASO-induced exon skipping within the transmembrane (16) or tyrosine kinase domains (18 and 21). In vivo ASO formulations induced >95% exon skipping in several models of nonsmall cell lung cancer (NSCLC) and were comparable in efficacy to erlotinib in reducing colony formation, cell viability, and migration in EGFR mutant NSCLC (PC9). However, unlike erlotinib, ASOs maintained their efficacy in both erlotinib-resistant subclones (PC9-GR) and wild-type overexpressing EGFR models (H292), in which erlotinib had no significant effect. The most dramatic ASO-induced phenotype resulted from targeting the EGFR kinase domain directly, which resulted in maximal inhibition of phosphorylation of EGFR, Akt, and Erk in both PC9 and PC9GR cells. Phosphoproteomic mass spectrometry confirmed highly congruent impacts of exon 16-, 18-, and 21-directed ASOs compared with erlotinib on PC9 genome-wide cell signaling. Furthermore, EGFR -directed ASOs had no impact in EGFR-independent NSCLC models, confirming an EGFR-specific therapeutic mechanism. Further exploration of synergy of ASOs with existing tyrosine kinase inhibitors may offer novel clinical models to improve EGFR-targeted therapies for both mutant and wild-type NSCLC patients.

In an ongoing, open-label, single-arm phase II study ( NCT02927301 ), 181 patients with untreated, resectable, stage IB-IIIB non-small cell lung cancer received two doses of neoadjuvant atezolizumab monotherapy. The primary end point was major pathological response (MPR; ≤10% viable malignant cells) in resected tumors without EGFR or ALK alterations. Of the 143 patients in the primary end point analysis, the MPR was 20% (95% confidence interval, 14-28%). With a minimum duration of follow-up of 3 years, the 3-year survival rate of 80% was encouraging. The most common adverse events during the neoadjuvant phase were fatigue (39%, 71 of 181) and procedural pain (29%, 53 of 181), along with expected immune-related toxicities; there were no unexpected safety signals. In exploratory analyses, MPR was predicted using the pre-treatment peripheral blood immunophenotype based on 14 immune cell subsets. Immune cell subsets predictive of MPR in the peripheral blood were also identified in the tumor microenvironment and were associated with MPR. This study of neoadjuvant atezolizumab in a large cohort of patients with resectable non-small cell lung cancer was safe and met its primary end point of MPR ≥ 15%. Data from this single-arm, non-randomized trial suggest that profiles of innate immune cells in pre-treatment peripheral blood may predict pathological response after neoadjuvant atezolizumab, but additional studies are needed to determine whether these profiles can inform patient selection and new therapeutic approaches., (© 2022. The Author(s).)

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment are often linked to drug resistance. Here, we found that coculture with CAFs or culture in CAF-conditioned medium unexpectedly induced drug sensitivity in certain lung cancer cell lines. Gene expression and secretome analyses of CAFs and normal lung-associated fibroblasts (NAFs) revealed differential abundance of insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), which promoted or inhibited, respectively, signaling by the receptor IGF1R and the kinase FAK. Similar drug sensitization was seen in gefitinib-resistant, EGFR -mutant PC9GR lung cancer cells treated with recombinant IGFBPs. Conversely, drug sensitivity was decreased by recombinant IGFs or conditioned medium from CAFs in which IGFBP5 or IGFBP6 was silenced. Phosphoproteomics and receptor tyrosine kinase (RTK) array analyses indicated that exposure of PC9GR cells to CAF-conditioned medium also inhibited compensatory IGF1R and FAK signaling induced by the EGFR inhibitor osimertinib. Combined small-molecule inhibition of IGF1R and FAK phenocopied the CAF-mediated effects in culture and increased the antitumor effect of osimertinib in mice. Cells that were osimertinib resistant and had MET amplification or showed epithelial-to-mesenchymal transition also displayed residual sensitivity to IGFBPs. Thus, CAFs promote or reduce drug resistance in a context-dependent manner, and deciphering the relationship between the differential content of CAF secretomes and the signaling dependencies of the tumor may reveal effective combination treatment strategies.

Purpose: The adenosine 2A receptor (A2AR) mediates the immunosuppressive effects of adenosine in the tumor microenvironment and is highly expressed in non-small cell lung cancer (NSCLC). Taminadenant (PBF509/NIR178) is an A2AR antagonist able to reactivate the antitumor immune response., Patients and Methods: In this phase I/Ib, dose-escalation/expansion study, patients with advanced/metastatic NSCLC and ≥1 prior therapy received taminadenant (80-640 mg, orally, twice a day) with or without spartalizumab (anti-programmed cell death-1, 400 mg, i.v., every 4 weeks). Primary endpoints were safety, tolerability, and feasibility of the combination., Results: During dose escalation, 25 patients each received taminadenant alone or with spartalizumab; 19 (76.0%) and 9 (36.0%) had received prior immunotherapy, respectively. Dose-limiting toxicities (all Grade 3) with taminadenant alone were alanine/aspartate aminotransferase increase and nausea [n = 1 (4.0%) each; 640 mg], and in the combination group were pneumonitis [n = 2 (8.0%); 160 and 240 mg] and fatigue and alanine/aspartate aminotransferase increase [n = 1 (4.0%) each; 320 mg]; pneumonitis cases responded to steroids rapidly and successfully. Complete and partial responses were observed in one patient each in the single-agent and combination groups; both were immunotherapy naïve. In the single-agent and combination groups, 7 and 14 patients experienced stable disease; 7 and 6 patients were immunotherapy pretreated, respectively., Conclusions: Taminadenant, with and without spartalizumab, was well tolerated in patients with advanced NSCLC. The maximum tolerated dose of taminadenant alone was 480 mg twice a day, and 240 mg twice a day plus spartalizumab. Efficacy was neither a primary or secondary endpoint; however, some clinical benefit was noted regardless of prior immunotherapy or programmed cell death ligand-1 status., (©2022 American Association for Cancer Research.)

To aid in the prioritization of deubiquitinases (DUBs) as anticancer targets, we developed an approach combining activity-based protein profiling (ABPP) with mass spectrometry in both non-small cell lung cancer (NSCLC) tumor tissues and cell lines along with analysis of available RNA interference and CRISPR screens. We identified 67 DUBs in NSCLC tissues, 17 of which were overexpressed in adenocarcinoma or squamous cell histologies and 12 of which scored as affecting lung cancer cell viability in RNAi or CRISPR screens. We used the CSN5 inhibitor, which targets COPS5/CSN5, as a tool to understand the biological significance of one of these 12 DUBs, COPS6, in lung cancer. Our study provides a powerful resource to interrogate the role of DUB signaling biology and nominates druggable targets for the treatment of lung cancer subtypes.

More than 50 years after the discovery of RAS family proteins, which harbor the most common activating mutations in cancer, the U.S. Food and Drug Administration approved the first direct allele-specific inhibitor of mutant KRAS in lung cancer. We highlight the history of discovering RAS and decades of studies targeting KRAS -driven lung cancer. A landmark article by Shokat and colleagues in 2013 elucidated allosteric inhibition of this undruggable target and paved the way for the first-in-class direct KRAS G12C inhibitor. Although these drugs have impressive 36%-45% objective response rates with a median duration of response of 10 months, many tumors do not respond, and diverse mechanisms of resistance have already been observed; this includes new KRAS alterations, activation of alternate RTK pathway proteins, bypass pathways, and transcriptional remodeling. These resistance mechanisms can be profiled using tissue-based and plasma-based testing and help to inform clinical trial options for patients. We conclude with a discussion of research informing ongoing clinical trials to rationally test promising treatments to thwart or overcome resistance to KRAS G12C inhibitors and target other KRAS -altered lung cancers.

To better understand the signaling complexity of AXL, a member of the tumor-associated macrophage (TAM) receptor tyrosine kinase family, we created a physical and functional map of AXL signaling interactions, phosphorylation events, and target-engagement of three AXL tyrosine kinase inhibitors (TKI). We assessed AXL protein complexes using proximity-dependent biotinylation (BioID), effects of AXL TKI on global phosphoproteins using mass spectrometry, and target engagement of AXL TKI using activity-based protein profiling. BioID identifies AXL-interacting proteins that are mostly involved in cell adhesion/migration. Global phosphoproteomics show that AXL inhibition decreases phosphorylation of peptides involved in phosphatidylinositol-mediated signaling and cell adhesion/migration. Comparison of three AXL inhibitors reveals that TKI RXDX-106 inhibits pAXL, pAKT, and migration/invasion of these cells without reducing their viability, while bemcentinib exerts AXL-independent phenotypic effects on viability. Proteomic characterization of these TKIs demonstrates that they inhibit diverse targets in addition to AXL, with bemcentinib having the most off-targets. AXL and EGFR TKI cotreatment did not reverse resistance in cell line models of erlotinib resistance. However, a unique vulnerability was identified in one resistant clone, wherein combination of bemcentinib and erlotinib inhibited cell viability and signaling. We also show that AXL is overexpressed in approximately 30% to 40% of nonsmall but rarely in small cell lung cancer. Cell lines have a wide range of AXL expression, with basal activation detected rarely., Implications: Our study defines mechanisms of action of AXL in lung cancers which can be used to establish assays to measure drug targetable active AXL complexes in patient tissues and inform the strategy for targeting it's signaling as an anticancer therapy., (©2022 American Association for Cancer Research.)

PARP inhibitors (PARPis) display single-agent anticancer activity in small cell lung cancer (SCLC) and other neuroendocrine tumors independent of BRCA1/2 mutations. Here, we determine the differential efficacy of multiple clinical PARPis in SCLC cells. Compared with the other PARPis rucaparib, olaparib, and niraparib, talazoparib displays the highest potency across SCLC, including SLFN11-negative cells. Chemical proteomics identifies PARP16 as a unique talazoparib target in addition to PARP1. Silencing PARP16 significantly reduces cell survival, particularly in combination with PARP1 inhibition. Drug combination screening reveals talazoparib synergy with the WEE1/PLK1 inhibitor adavosertib. Global phosphoproteomics identifies disparate effects on cell-cycle and DNA damage signaling thereby illustrating underlying mechanisms of synergy, which is more pronounced for talazoparib than olaparib. Notably, silencing PARP16 further reduces cell survival in combination with olaparib and adavosertib. Together, these data suggest that PARP16 contributes to talazoparib's overall mechanism of action and constitutes an actionable target in SCLC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A monocarboxylic inhibitor was designed and synthesized to disrupt the protein-protein interaction (PPI) between GRB2 and phosphotyrosine-containing proteins. Biochemical characterizations show compound 7 binds with the Src homology 2 (SH2) domain of GRB2 and is more potent than EGFR 1068 phosphopeptide 14-mer. X-ray crystallographic studies demonstrate compound 7 occupies the GRB2 binding site for phosphotyrosine-containing sequences and reveal key structural features for GRB2-inhibitor binding. This compound with a -1 formal charge offers a new direction for structural optimization to generate cell-permeable inhibitors for this key protein target of the aberrant Ras-MAPK signaling cascade., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: Non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor ( EGFR ) exon 20 insertion (Exon20ins) mutations exhibits inherent resistance to approved tyrosine kinase inhibitors. Amivantamab, an EGFR-MET bispecific antibody with immune cell-directing activity, binds to each receptor's extracellular domain, bypassing resistance at the tyrosine kinase inhibitor binding site., Methods: CHRYSALIS is a phase I, open-label, dose-escalation, and dose-expansion study, which included a population with EGFR Exon20ins NSCLC. The primary end points were dose-limiting toxicity and overall response rate. We report findings from the postplatinum EGFR Exon20ins NSCLC population treated at the recommended phase II dose of 1,050 mg amivantamab (1,400 mg, ≥ 80 kg) given once weekly for the first 4 weeks and then once every 2 weeks starting at week 5., Results: In the efficacy population (n = 81), the median age was 62 years (range, 42-84 years); 40 patients (49%) were Asian, and the median number of previous lines of therapy was two (range, 1-7). The overall response rate was 40% (95% CI, 29 to 51), including three complete responses, with a median duration of response of 11.1 months (95% CI, 6.9 to not reached). The median progression-free survival was 8.3 months (95% CI, 6.5 to 10.9). In the safety population (n = 114), the most common adverse events were rash in 98 patients (86%), infusion-related reactions in 75 (66%), and paronychia in 51 (45%). The most common grade 3-4 adverse events were hypokalemia in six patients (5%) and rash, pulmonary embolism, diarrhea, and neutropenia in four (4%) each. Treatment-related dose reductions and discontinuations were reported in 13% and 4% of patients, respectively., Conclusion: Amivantamab, via its novel mechanism of action, yielded robust and durable responses with tolerable safety in patients with EGFR Exon20ins mutations after progression on platinum-based chemotherapy., Competing Interests: Keunchil ParkConsulting or Advisory Role: AstraZeneca, Lilly, Ono Pharmaceutical, Bristol Myers Squibb, MSD, Blueprint Medicines, Amgen, Merck KGaA, LOXO, AbbVie, Daiichi Sankyo, Boehringer Ingelheim, JNJ, Eisai, Puma BiotechnologySpeakers' Bureau: Boehringer IngelheimResearch Funding: AstraZeneca, MSD Oncology Eric B. HauraConsulting or Advisory Role: Janssen Oncology, Revolution Medicines, Ellipses Pharma, Janssen Research & Development, AmgenResearch Funding: Janssen, Novartis, Revolution Medicines, AstraZeneca, Incyte, Genentech, Spectrum PharmaceuticalsPatents, Royalties, Other Intellectual Property: Protein-Protein Interactions as Biomarkers Patent Natasha LeighlHonoraria: Boehringer IngelheimConsulting or Advisory Role: XcoveryResearch Funding: Novartis, Roche Canada, Guardant Health, MSD, EMD Serono, LillyTravel, Accommodations, Expenses: Merck Sharp & Dohme, Bristol Myers Squibb, Nektar, GlaxoSmithKline, Roche, AstraZeneca Paul MitchellHonoraria: MSD, AstraZeneca, RocheConsulting or Advisory Role: Roche, Specialised Therapeutics, PUMA Biotechnology, AstraZeneca, Boehringer Ingelheim, BMS, MSD, Amgen Catherine A. ShuConsulting or Advisory Role: AstraZeneca, Boehringer Ingelheim, Genentech/RocheResearch Funding: Celgene, Genentech/Roche, Janssen, MedImmuneTravel, Accommodations, Expenses: Genentech/Roche Nicolas GirardEmployment: AstraZenecaConsulting or Advisory Role: Roche, Lilly, Boehringer Ingelheim, AstraZeneca, Novartis, Pfizer, Bristol Myers Squibb, MSD, Takeda, GlaxoSmithKline, AbbVie, Pharmamar, Janssen, SanofiResearch Funding: Roche, AstraZeneca, Boehringer IngelheimTravel, Accommodations, Expenses: Roche, AstraZeneca, Bristol Myers Squibb, MSD Oncology Santiago ViteriConsulting or Advisory Role: Roche, Bristol Myers Squibb, Janssen, Takeda, Reddy Pharma Iberia, Merck KGaASpeakers' Bureau: Bristol Myers Squibb, RocheTravel, Accommodations, Expenses: Roche, MSD, Merck KGaA Ji-Youn HanHonoraria: Roche, AstraZeneca, Bristol Myers Squibb, TakedaConsulting or Advisory Role: MSD Oncology, AstraZeneca, Bristol Myers Squibb, Lilly, Novartis, Takeda, PfizerResearch Funding: Roche, Pfizer, Ono Pharmaceutical, Takeda Chee Khoon LeeHonoraria: AstraZeneca, Pfizer, Amgen, Takeda, Yuhan, Boehringer IngelheimConsulting or Advisory Role: Novartis, Boehringer Ingelheim, Takeda, AstraZeneca, Yuhan, AmgenResearch Funding: AstraZeneca, Roche, Merck KGaA Joshua K. SabariConsulting or Advisory Role: AstraZeneca, Janssen Oncology, Navire, Pfizer, Regeneron, Medscape, Takeda Alexander I. SpiraStock and Other Ownership Interests: LillyHonoraria: CytomX Therapeutics, AstraZeneca/MedImmune, Merck, Takeda, Amgen, Janssen Oncology, Novartis, Bristol Myers Squibb, BayerConsulting or Advisory Role: Array BioPharma, Incyte, Amgen, Novartis, AstraZeneca/MedImmune, Mirati Therapeutics, Gritstone Oncology, Jazz Pharmaceuticals, Merck, Bristol Myers SquibbResearch Funding: Roche, AstraZeneca, Boehringer Ingelheim, Astellas Pharma, MedImmune, Novartis, Newlink Genetics, Incyte, AbbVie, Ignyta, LAM Therapeutics, Trovagene, Takeda, Macrogenics, CytomX Therapeutics, LAM Therapeutics, Astex Pharmaceuticals, Bristol Myers Squibb, Loxo, Arch Therapeutics, Gritstone Oncology, Plexxikon, Amgen, Loxo, Daiichi Sankyo, ADC Therapeutics, Janssen Oncology, Mirati Therapeutics, Rubius Therapeutics Dong-Wan KimResearch Funding: Alpha Biopharma, AstraZeneca/MedImmune, Hanmi, Janssen, Merus, Mirati Therapeutics, MSD, Novartis, Ono Pharmaceutical, Pfizer, Roche/Genentech, Takeda, TP Therapeutics, Xcovery, Yuhan, Boehringer Ingelheim, Amgen, Daiichi Sankyo, Chong Kun Dang Pharmaceutical, BridgeBio Pharma, GlaxoSmithKlineTravel, Accommodations, Expenses: Daiichi Sankyo, Amgen Ki Hyeong LeeConsulting or Advisory Role: Bristol Myers Squibb, MSD, AstraZeneca, Pfizer, Lilly Rachel E. SanbornHonoraria: AstraZeneca, AmgenConsulting or Advisory Role: Genentech/Roche, AstraZeneca, EMD Serono, Blueprint Medicines, Daiichi Sankyo/Lilly, Janssen Oncology, MacrogenicsResearch Funding: Bristol Myers Squibb, MedImmune, Merck, AstraZenecaTravel, Accommodations, Expenses: AstraZeneca José TrigoConsulting or Advisory Role: Takeda, Boehringer Ingelheim, Bristol Myers Squibb, Merck SeronoSpeakers' Bureau: MSD Oncology, AstraZeneca, Merck Serono, RocheTravel, Accommodations, Expenses: MSD Oncology, Bristol Myers Squibb, AstraZeneca Koichi GotoHonoraria: Guardant Health, Janssen, Daiichi Sankyo Co Ltd, Amgen, Eisai, Kyowa Kirin Co Ltd, Bristol Myers Squibb, AstraZeneca Japan, Pfizer, Chugai Pharma, Taiho Pharmaceutical, Ono Pharmaceutical, Novartis, Lilly Japan, Boehringer Ingelheim, Life Technologies, MSD K.K, Nippon Kayaku, Takeda, OtsukaConsulting or Advisory Role: OtsukaResearch Funding: Medical & Biological Laboratories Co Ltd, Kyowa Kirin Co Ltd, Amgen Astellas BioPharma, Kissei Pharmaceutical, Merck, Merus, NEC Corporation, Spectrum Pharmaceuticals, Shanghai HaiHe Pharmaceutical, MSD K.K, AstraZeneca Japan, Taiho Pharmaceutical, Chugai Pharma, Boehringer Ingelheim, Ono Pharmaceutical, Sumitomo Dainippon Pharma Co Ltd, Takeda, Astellas Pharma, Eisai, Lilly Japan, Pfizer, Bristol Myers Squibb, Ignyta, Life Technologies, Janssen, Xcoo, Loxo, Sysmex, Amgen James Chih-Hsin YangHonoraria: Boehringer Ingelheim, Roche, MSD, AstraZeneca, Novartis, Bristol Myers Squibb, Ono Pharmaceutical, Takeda, Lilly, PfizerConsulting or Advisory Role: Boehringer Ingelheim, Novartis, AstraZeneca, Clovis Oncology, Lilly, MSD Oncology, Merck Serono, Celgene, Bayer, Pfizer, Ono Pharmaceutical, Bristol Myers Squibb, Yuhan, Hansoh, Blueprint Medicines, Daiichi Sankyo, G1 Therapeutics, AbbVie, Takeda Oncology, Amgen, Incyte, Merck Serono, Jenssen, GSK, Takeda, Daiichi Sankyo, NovartisTravel, Accommodations, Expenses: Pfizer Ramaswamy GovindanHonoraria: Genentech/AbbVie, AbbVie, Geneplus-Beijing InstituteConsulting or Advisory Role: Genentech/Roche, AbbVie, AstraZeneca/MedImmune, Pfizer, Bristol Myers Squibb, Nektar, Jounce Therapeutics, F. Hoffmann La Roche AG, Janssen, Amgen, Achilles Therapeutics Joshua M. BaumlConsulting or Advisory Role: Bristol Myers Squibb, Merck, AstraZeneca, Genentech, Celgene, Boehringer Ingelheim, Guardant Health, Takeda, Novartis, Janssen, Ayala Pharmaceuticals, Regeneron, Inivata, Novartis, Foundation MedicineResearch Funding: Merck, Carevive Systems, Novartis, Incyte, Bayer, Janssen, AstraZeneca, Takeda, Amgen, Pfizer, Mirati Therapeutics Pilar GarridoConsulting or Advisory Role: Roche Pharma AG, AstraZeneca, MSD Oncology, Bristol Myers Squibb, Takeda, Lilly, Pfizer, Novartis/Pfizer, Boehringer Ingelheim, AbbVie, Amgen, Bayer, Gebro Pharma, Nordic Group, Boehringer Ingelheim, Janssen Biotech, Janssen Oncology, GlaxoSmithKlineSpeakers' Bureau: Roche Pharma AG, Takeda, AstraZeneca, MSD Oncology, BMS, Pfizer, Novartis, Boehringer Ingelheim, Nordic Group, Janssen, Boehringer Ingelheim, Janssen OncologyTravel, Accommodations, Expenses: AstraZeneca, Roche, Bristol Myers SquibbOther Relationship: Janssen Oncology, Novartis Matthew G. KrebsHonoraria: RocheConsulting or Advisory Role: Roche, Achilles Therapeutics, Janssen, Seattle Genetics, OM Pharma, BayerSpeakers' Bureau: Roche, JanssenResearch Funding: RocheTravel, Accommodations, Expenses: AstraZeneca, BerGenBio, Immutep Karen L. ReckampConsulting or Advisory Role: Amgen, Tesaro, Boehringer Ingelheim, Takeda, AstraZeneca, Seattle Genetics, Calithera Biosciences, Genentech, Blueprint Medicines, Daiichi Sankyo/Lilly, EMD Serono, Janssen Oncology, Lilly, Merck KGaAResearch Funding: Bristol Myers Squibb, Pfizer, ARIAD, Xcovery, Adaptimmune, Genentech/Roche, Boehringer Ingelheim, AbbVie, ACEA Biosciences, Loxo, GlaxoSmithKline, Guardant Health, Janssen Oncology, Seattle Genetics, Zeno Pharmaceuticals, Calithera Biosciences, Elevation Oncology, Daiichi Sankyo/Astra Zeneca John XieEmployment: JNJStock and Other Ownership Interests: JNJ Joshua C. CurtinEmployment: Janssen Research & DevelopmentStock and Other Ownership Interests: Johnson & Johnson/Janssen Nahor Haddish-BerhaneEmployment: Johnson & JohnsonStock and Other Ownership Interests: Johnson & Johnson Amy RoshakEmployment: Janssen Pharmaceutical Company of Johnson & JohnsonStock and Other Ownership Interests: Johnson & Johnson Dawn MillingtonEmployment: Janssen Research & DevelopmentStock and Other Ownership Interests: Johnson & Johnson/JanssenTravel, Accommodations, Expenses: Janssen Research & Development Patricia LorenziniEmployment: Janssen Pharmaceuticals of Johnson & Johnson Meena ThayuEmployment: Janssen OncologyStock and Other Ownership Interests: Johnson & Johnson/JanssenOther Relationship: Janssen Oncology Roland E. KnoblauchEmployment: Johnson & JohnsonStock and Other Ownership Interests: Johnson & JohnsonTravel, Accommodations, Expenses: Johnson & Johnson Byoung Chul ChoLeadership: Gencurix Inc, Interpark Bio Convergence CorpStock and Other Ownership Interests: TheraCanVac Inc, Gencurix Inc, Bridgebio Therapeutics, KANAPH Therapeutic Inc, Cyrus Therapeutics, Interpark Bio Convergence CorpConsulting or Advisory Role: Novartis, AstraZeneca, Boehringer Ingelheim, Roche, Bristol Myers Squibb, Yuhan, Pfizer, Janssen, Takeda, MSD, Ono Pharmaceutical, Eli Lilly, Medpacto, Blueprint Medicines, KANAPH Therapeutic Inc, Brigebio Therapeutics, Cyrus Therapeutics, Guardant Health, Oscotec IncResearch Funding: Novartis, Bayer, AstraZeneca, MOGAM Institute, Dong-A ST, Champions Oncology, Janssen, Yuhan, Ono, Dizal Pharma, MSD, AbbVie, Medpacto, GIInnovation, Eli Lilly, Blueprint Medicines, Interpark Bio Convergence CorpPatents, Royalties, Other Intellectual Property: Champions OncologyOther Relationship: DAAN BiotherapeuticsNo other potential conflicts of interest were reported.

Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown activity in melanoma, but has not been previously evaluated in metastatic non-small cell lung cancer. We conducted a single-arm open-label phase 1 trial ( NCT03215810 ) of TILs administered with nivolumab in 20 patients with advanced non-small cell lung cancer following initial progression on nivolumab monotherapy. The primary end point was safety and secondary end points included objective response rate, duration of response and T cell persistence. Autologous TILs were expanded ex vivo from minced tumors cultured with interleukin-2. Patients received cyclophosphamide and fludarabine lymphodepletion, TIL infusion and interleukin-2, followed by maintenance nivolumab. The end point of safety was met according to the prespecified criteria of ≤17% rate of severe toxicity (95% confidence interval, 3-29%). Of 13 evaluable patients, 3 had confirmed responses and 11 had reduction in tumor burden, with a median best change of 35%. Two patients achieved complete responses that were ongoing 1.5 years later. In exploratory analyses, we found T cells recognizing multiple types of cancer mutations were detected after TIL treatment and were enriched in responding patients. Neoantigen-reactive T cell clonotypes increased and persisted in peripheral blood after treatment. Cell therapy with autologous TILs is generally safe and clinically active and may constitute a new treatment strategy in metastatic lung cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Human tissue samples commonly preserved as formalin-fixed paraffin-embedded (FFPE) tissues after diagnostic or surgical procedures in the clinic represent an invaluable source of clinical specimens for in-depth characterization of signaling networks to assess therapeutic options. Tyrosine phosphorylation (pTyr) plays a fundamental role in cellular processes and is commonly dysregulated in cancer but has not been studied to date in FFPE samples. In addition, pTyr analysis that may otherwise inform therapeutic interventions for patients has been limited by the requirement for large amounts of frozen tissue. Here we describe a method for highly sensitive, quantitative analysis of pTyr signaling networks, with hundreds of sites quantified from one to two 10-μm sections of FFPE tissue specimens. A combination of optimized magnetic bead-based sample processing, optimized pTyr enrichment strategies, and tandem mass tag multiplexing enabled in-depth coverage of pTyr signaling networks from small amounts of input material. Phosphotyrosine profiles of flash-frozen and FFPE tissues derived from the same tumors suggested that FFPE tissues preserve pTyr signaling characteristics in patient-derived xenografts and archived clinical specimens. pTyr analysis of FFPE tissue sections from breast cancer tumors as well as lung cancer tumors highlighted patient-specific oncogenic driving kinases, indicating potential targeted therapies for each patient. These data suggest the capability for direct translational insight from pTyr analysis of small amounts of FFPE tumor tissue specimens. SIGNIFICANCE: This study reports a highly sensitive method utilizing FFPE tissues to identify dysregulated signaling networks in patient tumors, opening the door for direct translational insights from FFPE tumor tissue banks in hospitals., (©2021 The Authors; Published by the American Association for Cancer Research.)

Rationale: Recent studies have discovered several unique tumor response subgroups outside of response classification by Response Evaluation Criteria for Solid Tumors (RECIST), such as mixed response and oligometastasis. These subtypes have a distinctive property, lesion heterogeneity defined as diversity of tumor growth profiles in RECIST target lesions. Furthermore, many cancer clinical trials have been activated to evaluate various treatment options for heterogeneity-related subgroups (e.g., 29 trials so far listed in clinicaltrials.gov for cancer patients with oligometastasis). Some of the trials have shown survival benefit by tailored treatment strategies. This evidence presents the unmet need to incorporate lesion heterogeneity to improve RECIST response classification., Method: An approach for Lesion Heterogeneity Classification (LeHeC) was developed using a contemporary statistical approach to assess target lesion variation, characterize patient treatment response, and translate informative evidence to improving treatment strategy. A mixed effect linear model was used to determine lesion heterogeneity. Further analysis was conducted to classify various types of lesion variation and incorporate with RECIST to enhance response classification. A study cohort of 110 target lesions from 36 lung cancer patients was used for evaluation., Results: Due to small sample size issue, the result was exploratory in nature. By analyzing RECIST target lesion data, the LeHeC approach detected a high prevalence (n = 21; 58%) of lesion heterogeneity. Subgroup classification revealed several informative distinct subsets in a descending order of lesion heterogeneity: mix of progression and regression (n = 7), mix of progression and stability (n = 9), mix of regression and stability (n = 5), and non-heterogeneity (n = 15). Evaluation for association of lesion heterogeneity and RECIST best response classification showed lesion heterogeneity commonly occurred in each response group (stable disease: 16/27; 59%; partial response: 3/5; 60%; progression disease: 2/4; 50%). Survival analysis showed a differential trend of overall survival between heterogeneity and non-heterogeneity in RECIST response groups., Conclusion: This is the first study to evaluate lesion heterogeneity, an underappreciated metric, for RECIST application in oncology clinical trials. Results indicated lesion heterogeneity is not an uncommon event. The LeHeC approach could enhance RECIST response classification by utilizing granular lesion level discovery of heterogeneity., Competing Interests: The authors have read the journal’s policy and have the following competing interests: AC serves on Advisory Boards and/or Speaker Bureaus for Novartis, Merck, Genentech, Takeda, Jazz, Blueprint, Amgen and Pfizer and has research support from Bristol-Myers Squibb, and AstraZeneca. BC is an advisor or consultant with E.R. Squibb & Sons LLC, Celgene Corp, Gilead Sciences Inc., Achilles plc., Xilio Therapeutics, and KSQ Therapeutics. He is on the Speaker’s Bureau for Hoffman-LaRoche AG, E.R. Squibb & Sons LLC, AstraZeneca LLC. He has research support from Prometheus Inc., Iovance Biotherapeutics Inc., Adaptive Biotechnologies Corp. JEG serves in Advisory Board or consultant, or has research support from AstraZeneca, Blueprint Medicines, Boehringer Ingelheim, Bristol-Myers Squibb, EMD Serono - Merck KGaA, Genetech, G 1 Therapeutics, Inivata, Merck, Novartis, Pfizer, and Ludwig Institute of Cancer Research. EH is advisory board or consultant for Ellipses, Janssen, Amgen and Revolution. AS has travel reimbursement or research support from Novartis, Daiichi Sankyo, Mersana, Genmab, AstraZeneca, and Turning Point Therapeutics, and Prime Oncology. AAB has research support from Bristol-Myers -Squib and serves on the advisory board of Memgen Inc. DTC has research from Bristol-Myers -Squib and Oncologies. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Purpose: Covalent inhibitors of KRAS G12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRAS G12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear., Experimental Design: We performed mass spectrometry-based phosphoproteomics analysis in KRAS G12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type-specific markers, we compared proteome and phosphoproteomes of KRAS G12C cells. Gene expression patterns of KRAS G12C cell lines and lung tumor tissues were examined., Results: Our analysis suggests cell type-specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRAS G12C , we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures., Conclusions: Our phosphoproteomic study identified cell type-adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi., (©2021 American Association for Cancer Research.)

Purpose: Osimertinib is an effective therapy in EGFR -mutant non-small cell lung cancer (NSCLC), but resistance invariably develops. Navitoclax is an oral inhibitor of BCL-2/BCL-xL that has exhibited synergy with osimertinib in preclinical models of EGFR -mutant NSCLC. In hematologic malignancies, BCL-2 family inhibitors in combination therapy effectively increase cellular apoptosis and decrease drug resistance., Patients and Methods: This single-arm phase Ib study evaluated safety, tolerability, and feasibility of osimertinib and navitoclax, including dose expansion in T790M-positive patients at the recommended phase II dose (RP2D). Eligible patients had advanced EGFR -mutant NSCLC with prior tyrosine kinase inhibitor exposure. Five dose levels were planned with osimertinib from 40 to 80 mg orally daily and navitoclax from 150 to 325 mg orally daily., Results: A total of 27 patients were enrolled (18 in the dose-escalation cohort and nine in the dose-expansion cohort): median age 65, 67% female, 48% exon 19 del, and 37% L858R, median one prior line of therapy. The most common adverse events were lymphopenia (37%), fatigue (22%), nausea (22%), and thrombocytopenia (37%). No dose-limiting toxicities were seen in dose-escalation cohort; osimertinib 80 mg, navitoclax 150 mg was chosen as the RP2D. Most patients (78%) received >95% of planned doses through three cycles. In expansion cohort, objective response rate was 100% and median progression-free survival was 16.8 months. A proapoptotic effect from navitoclax was demonstrated by early-onset thrombocytopenia., Conclusions: Oral combination therapy with navitoclax and osimertinib was safe and feasible at RP2D with clinical efficacy. Early thrombocytopenia was common, supporting an target engagement by navitoclax. Further study of BCL-2/BCL-xL inhibition to enhance osimertinib activity is warranted., (©2020 American Association for Cancer Research.)

Highly collaborative scientists are often called on to extend their expertise to different types of projects and to expand the scope and scale of projects well beyond their previous experience. For a large-scale project involving "big data" to be successful, several different aspects of the research plan need to be developed and tested, which include but are not limited to the experimental design, sample collection, sample preparation, metadata recording, technical capability, data acquisition, approaches for data analysis, methods for integration of different data types, recruitment of additional expertise as needed to guide the project, and strategies for clear communication throughout the project. To capture this process, we describe an example project in proteogenomics that built on our collective expertise and experience. Key steps included definition of hypotheses, identification of an appropriate clinical cohort, pilot projects to assess feasibility, refinement of experimental designs, and extensive discussions involving the research team throughout the process. The goal of this chapter is to provide the reader with a set of guidelines to support development of other large-scale multiomics projects.

Analysis of tyrosine kinase signaling is critical for the development of targeted cancer therapy. Currently, immunoprecipitation of phosphotyrosine (pY) peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to profile tyrosine kinase substrates. A typical protocol requests 10 mg of total protein from ≈10 8 cells or 50-100 mg of tissue. Large sample requirements can be cost prohibitive or not feasible for certain experiments. Sample multiplexing using chemical labeling reduces the protein amount required for each sample, and newer approaches use a material-rich reference channel as a calibrator to trigger detection and quantification for smaller samples. Here, it is demonstrated that the tandem mass tag (TMT) calibrator approach reduces the sample input for pY profiling tenfold (to ≈1 mg total protein per sample from 10 7 cells grown in one plate), while maintaining the depth of pY proteome sampling and the biological content of the experiment. Data are available through PRIDE (PXD019764 for label-free and PXD018952 for TMT). This strategy opens more opportunities for pY profiling of large sample cohorts and samples with limited protein quantity such as immune cells, xenograft models, and human tumors., (© 2020 Wiley-VCH GmbH.)

Background: Abstaining from smoking after a cancer diagnosis is critical to mitigating the risk of multiple adverse health outcomes. Although many patients with cancer attempt to quit smoking, the majority relapse. The current randomized controlled trial evaluated the efficacy of adapting an evidence-based smoking relapse prevention (SRP) intervention for patients with cancer., Methods: The trial enrolled 412 patients newly diagnosed with cancer who had recently quit smoking. Participants were randomized to usual care (UC) or SRP. Participants in the UC group received the institution's standard of care for treating tobacco use. Participants in the SRP group in addition received a targeted educational DVD plus a validated self-help intervention for preventing smoking relapse. The primary outcome was smoking abstinence at 2 months, 6 months, and 12 months., Results: Abstinence rates for participants in the SRP and UC groups were 75% versus 71% at 2 months and 69% versus 64% at 6 months (Ps > .20). At 12 months, abstinence rates among survivors were 68% for those in the SRP group and 63% for those in the UC group (P = .38). Post hoc analyses revealed that across 2 months and 6 months, patients who were married/partnered were more likely to be abstinent after SRP than UC (P = .03)., Conclusions: A smoking relapse prevention intervention did not reduce relapse rates overall, but did appear to have benefited those participants who had the social support of a partner. Future work is needed to extend this effect to the larger population of patients., (© 2020 American Cancer Society.)