Novel CDK12/13 Inhibitors AU-15506 and AU-16770 Are Potent Anti-Cancer Agents in EGFR Mutant Lung Adenocarcinoma with and without Osimertinib Resistance.

Osimertinib is a third-generation epidermal growth factor receptor and tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of lung adenocarcinoma patients harboring EGFR mutations. However, acquired resistance to this targeted therapy is inevitable, leading to disease relapse within a few years. Therefore, understanding the molecular mechanisms of osimertinib resistance and identifying novel targets to overcome such resistance are unmet needs of cancer patients. Here, we investigated the efficacy of two novel CDK12/13 inhibitors, AU-15506 and AU-16770, in osimertinib-resistant EGFR mutant lung adenocarcinoma cells in culture and xenograft models in vivo. We demonstrate that these drugs, either alone or in combination with osimertinib, are potent inhibitors of osimertinib-resistant as well as -sensitive lung adenocarcinoma cells in culture. Interestingly, only the CDK12/13 inhibitor in combination with osimertinib, although not as monotherapy, suppresses the growth of resistant tumors in xenograft models in vivo. Taken together, the results of this study suggest that inhibition of CDK12/13 in combination with osimertinib has the potential to overcome osimertinib resistance in EGFR mutant lung adenocarcinoma patients.

Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors.

Mesothelin targeting CAR T cells have limited activity in patients. In this study, we sought to determine if efficacy of anti-mesothelin CAR T cells is dependent on the mesothelin epitopes that are recognized by them. To do so, we developed hYP218 (against membrane-proximal epitope) and SS1 (against membrane-distal epitope) CAR T cells. Their efficacy was assessed in vitro using mesothelin-positive tumor cell lines and in vivo in NSG mice with mesothelin-expressing ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived (NCI-Meso63) tumor xenografts. Persistence and tumor infiltration of CAR T cells was determined using flow cytometry. hYP218 CAR T cells killed cancer cells more efficiently than SS1 CAR T cells, with a two- to fourfold lower ET50 value (effector-to-target ratio for 50% killing of tumor cells). In mice with established tumors, single intravenous administration of hYP218 CAR T cells lead to improved tumor response and survival compared with SS1 CAR T cells, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared with SS1 CAR T cells, there was increased peripheral blood expansion, persistence, and tumor infiltration of hYP218 CAR T cells in the KLM-1 tumor model. Persistence of hYP218 CAR T cells in treated mice led to antitumor immunity when rechallenged with KLM-1 tumor cells. Our results show that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin-positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin-expressing cancers.

Alterations in HLA Class I-Presented Immunopeptidome and Class I-Interactome upon Osimertinib Resistance in EGFR Mutant Lung Adenocarcinoma.

Immune checkpoint inhibitor (ICI) therapy has been a paradigm shift in the treatment of cancer. ICI therapy results in durable responses and survival benefit for a large number of tumor types. Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has shown great efficacy treating EGFR mutant lung cancers; however, all patients eventually develop resistance. ICI therapy has not benefitted EGFR mutant lung cancer. Herein, we employed stable isotope labeling by amino acids in cell culture (SILAC) quantitative mass spectrometry-based proteomics to investigate potential immune escape molecular mechanisms in osimertinib resistant EGFR mutant lung adenocarcinoma by interrogating the alterations in the human leukocyte antigen (HLA) Class I-presented immunopeptidome, Class I-interactome, and the whole cell proteome between isogenic osimertinib-sensitive and -resistant human lung adenocarcinoma cells. Our study demonstrates an overall reduction in HLA class I-presented immunopeptidome and downregulation of antigen presentation core complex (e.g., TAP1 and ERAP1/2) and immunoproteasome in osimertinib resistant lung adenocarcinoma cells. Several key components in autophagy pathway are differentially altered. S100 proteins and SLC3A2 may play critical roles in reduced antigen presentation. Our dataset also includes ~1000 novel HLA class I interaction partners and hundreds of Class I-presented immunopeptides in EGFR mutant lung adenocarcinoma. This large-scale unbiased proteomics study provides novel insights and potential mechanisms of immune evasion of EGFR mutant lung adenocarcinoma.

SCAMP3 is a mutant EGFR phosphorylation target and a tumor suppressor in lung adenocarcinoma.

Mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain constitutively activate EGFR resulting in lung tumorigenesis. Activated EGFR modulates downstream signaling by altering phosphorylation-driven interactions that promote growth and survival. Secretory carrier membrane proteins (SCAMPs) are a family of transmembrane proteins that regulate recycling of receptor proteins, including EGFR. The potential role of SCAMPs in mutant EGFR function and tumorigenesis has not been elucidated. Using quantitative mass-spectrometry-based phosphoproteomics, we identified SCAMP3 as a target of mutant EGFRs in lung adenocarcinoma and sought to further investigate the role of SCAMP3 in the regulation of lung tumorigenesis. Here we show that activated EGFR, either directly or indirectly phosphorylates SCAMP3 at Y86 and this phosphorylation increases the interaction of SCAMP3 with both wild-type and mutant EGFRs. SCAMP3 knockdown increases lung adenocarcinoma cell survival and increases xenograft tumor growth in vivo, demonstrating a tumor suppressor role of SCAMP3 in lung tumorigenesis. The tumor suppressor function is a result of SCAMP3 promoting EGFR degradation and attenuating MAP kinase signaling pathways. SCAMP3 knockdown also increases multinucleated cells in culture, suggesting that SCAMP3 is required for efficient cytokinesis. The enhanced growth, increased colony formation, reduced EGFR degradation and multinucleation phenotype of SCAMP3-depleted cells were reversed by re-expression of wild-type SCAMP3, but not SCAMP3 Y86F, suggesting that Y86 phosphorylation is critical for SCAMP3 function. Taken together, the results of this study demonstrate that SCAMP3 functions as a novel tumor suppressor in lung cancer by modulating EGFR signaling and cytokinesis that is partly Y86 phosphorylation-dependent.

Clonal Evolution and Heterogeneity of Osimertinib Acquired Resistance Mechanisms in EGFR Mutant Lung Cancer.

Clonal evolution of osimertinib-resistance mechanisms in EGFR mutant lung adenocarcinoma is poorly understood. Using multi-region whole-exome and RNA sequencing of prospectively collected pre- and post-osimertinib-resistant tumors, including at rapid autopsies, we identify a likely mechanism driving osimertinib resistance in all patients analyzed. The majority of patients acquire two or more resistance mechanisms either concurrently or in temporal sequence. Focal copy-number amplifications occur subclonally and are spatially and temporally separated from common resistance mutations such as EGFR C797S. MET amplification occurs in 66% (n = 6/9) of first-line osimertinib-treated patients, albeit spatially heterogeneous, often co-occurs with additional acquired focal copy-number amplifications and is associated with early progression. Noteworthy osimertinib-resistance mechanisms discovered include neuroendocrine differentiation without histologic transformation, PD-L1, KRAS amplification, and ESR1-AKAP12, MKRN1-BRAF fusions. The subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance will likely require targeting multiple resistance mechanisms by combination therapies.

APOBEC Mutagenesis and Copy-Number Alterations Are Drivers of Proteogenomic Tumor Evolution and Heterogeneity in Metastatic Thoracic Tumors.

Intratumor mutational heterogeneity has been documented in primary non-small-cell lung cancer. Here, we elucidate mechanisms of tumor evolution and heterogeneity in metastatic thoracic tumors (lung adenocarcinoma and thymic carcinoma) using whole-exome and transcriptome sequencing, SNP array for copy-number alterations (CNAs), and mass-spectrometry-based quantitative proteomics of metastases obtained by rapid autopsy. APOBEC mutagenesis, promoted by increased expression of APOBEC3 region transcripts and associated with a high-risk APOBEC3 germline variant, correlated with mutational tumor heterogeneity. TP53 mutation status was associated with APOBEC hypermutator status. Interferon pathways were enriched in tumors with high APOBEC mutagenesis and IFN-γ-induced expression of APOBEC3B in lung adenocarcinoma cells, suggesting that the immune microenvironment may promote mutational heterogeneity. CNAs occurring late in tumor evolution correlated with downstream transcriptomic and proteomic heterogeneity, although global proteomic heterogeneity was significantly greater than transcriptomic and CNA heterogeneity. These results illustrate key mechanisms underlying multi-dimensional heterogeneity in metastatic thoracic tumors.

Quantitative Mass Spectrometry to Interrogate Proteomic Heterogeneity in Metastatic Lung Adenocarcinoma and Validate a Novel Somatic Mutation CDK12-G879V.

Lung cancer is the leading cause of cancer death in both men and women. Tumor heterogeneity is an impediment to targeted treatment of all cancers, including lung cancer. Here, we sought to characterize tumor proteome and phosphoproteome changes by longitudinal, prospective collection of tumor tissue from an exceptional responder lung adenocarcinoma patient who survived with metastatic lung adenocarcinoma for over seven years while undergoing HER2-directed therapy in combination with chemotherapy. We employed "Super-SILAC" and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and eight distinct metastatic progressive lymph nodes collected during these seven years, including five lymph nodes procured at autopsy. We identified specific signaling networks enriched in lung compared with the lymph node metastatic sites. We correlated the changes in protein abundance with changes in copy number alteration (CNA) and transcript expression. ERBB2/HER2 protein expression was higher in lung, consistent with a higher degree of ERBB2 amplification in lung compared with the lymph node metastatic sites. To further interrogate the mass spectrometry data, a patient-specific database was built by incorporating all the somatic and germline variants identified by whole genome sequencing (WGS) of genomic DNA from the lung, one lymph node metastatic site and blood. An extensive validation pipeline was built to confirm variant peptides. We validated 360 spectra corresponding to 55 germline and 6 somatic variant peptides. Targeted MRM assays revealed two novel variant somatic peptides, CDK12-G879V and FASN-R1439Q, expressed in lung and lymph node metastatic sites, respectively. The CDK12-G879V mutation likely results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites. Knockdown of CDK12 in lung adenocarcinoma cells increased chemotherapy sensitivity which was rescued by wild type, but not CDK12-G879V expression, consistent with the complete resolution of the lung metastatic sites in this patient.

Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.

Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747LREA750, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238/Y1239 and MET-Y1252/1253. This study provides unique insight into the TKI-mediated modulation of mutant EGFR signaling, which can be applied to the development of biomarkers of EGFR TKI response.

Genomic profiling of multiple sequentially acquired tumor metastatic sites from an "exceptional responder" lung adenocarcinoma patient reveals extensive genomic heterogeneity and novel somatic variants driving treatment response.

We used next-generation sequencing to identify somatic alterations in multiple metastatic sites from an "exceptional responder" lung adenocarcinoma patient during his 7-yr course of ERBB2-directed therapies. The degree of heterogeneity was unprecedented, with ∼1% similarity between somatic alterations of the lung and lymph nodes. One novel translocation, PLAG1-ACTA2, present in both sites, up-regulated ACTA2 expression. ERBB2, the predominant driver oncogene, was amplified in both sites, more pronounced in the lung, and harbored an L869R mutation in the lymph node. Functional studies showed increased proliferation, migration, metastasis, and resistance to ERBB2-directed therapy because of L869R mutation and increased migration because of ACTA2 overexpression. Within the lung, a nonfunctional CDK12, due to a novel G879V mutation, correlated with down-regulation of DNA damage response genes, causing genomic instability, and sensitivity to chemotherapy. We propose a model whereby a subclone metastasized early from the primary site and evolved independently in lymph nodes.

EGFR-targeted therapy results in dramatic early lung tumor regression accompanied by imaging response and immune infiltration in EGFR mutant transgenic mouse models.

Lung adenocarcinoma patients harboring kinase domain mutations in Epidermal growth factor receptor (EGFR) have significant clinical benefit from EGFR-targeted tyrosine kinase inhibitors (TKIs). Although a majority of patients experience clinical symptomatic benefit immediately, an objective response can only be demonstrated after 6-8 weeks of treatment. Evaluation of patient response by imaging shows that 30-40% of patients do not respond due to intrinsic resistance to these TKIs. We investigated immediate-early effects of EGFR-TKI treatment in mutant EGFR-driven transgenic mouse models by FDG-PET and MRI and correlated the effects on the tumor and the tumor microenvironment. Within 24 hours of erlotinib treatment we saw approximately 65% tumor regression in mice with TKI-sensitive EGFRL858R lung adenocarcinoma. However, mice with EGFRL858R/T790M-driven tumors did not respond to either erlotinib or afatinib monotherapy, but did show a significant tumor response to afatinib-cetuximab combination treatment. The imaging responses correlated with the inhibition of downstream EGFR signaling, increased apoptosis, and decreased proliferation in the tumor tissues. In EGFRL858R-driven tumors, we saw a significant increase in CD45+ leukocytes, NK cells, dendritic cells, macrophages and lymphocytes, particularly CD8+ T cells. In response to erlotinib, these dendritic cells and macrophages had significantly higher MHC class II expression, indicating increased antigen-presenting capabilities. Together, results of our study provide novel insight into the immediate-early therapeutic response to EGFR TKIs in vivo.

Loss of MIG6 Accelerates Initiation and Progression of Mutant Epidermal Growth Factor Receptor-Driven Lung Adenocarcinoma.

UNLABELLED: Somatic mutations in the EGFR kinase domain drive lung adenocarcinoma. We have previously identified MIG6, an inhibitor of ERBB signaling and a potential tumor suppressor, as a target for phosphorylation by mutant EGFRs. Here, we demonstrate that MIG6 is a tumor suppressor for the initiation and progression of mutant EGFR-driven lung adenocarcinoma in mouse models. Mutant EGFR-induced lung tumor formation was accelerated in Mig6-deficient mice, even with Mig6 haploinsufficiency. We demonstrate that constitutive phosphorylation of MIG6 at Y394/Y395 in EGFR-mutant human lung adenocarcinoma cell lines is associated with an increased interaction of MIG6 with mutant EGFR, which may stabilize EGFR protein. MIG6 also fails to promote mutant EGFR degradation. We propose a model whereby increased tyrosine phosphorylation of MIG6 decreases its capacity to inhibit mutant EGFR. Nonetheless, the residual inhibition is sufficient for MIG6 to delay mutant EGFR-driven tumor initiation and progression in mouse models. SIGNIFICANCE: This study demonstrates that MIG6 is a potent tumor suppressor for mutant EGFR-driven lung tumor initiation and progression in mice and provides a possible mechanism by which mutant EGFR can partially circumvent this tumor suppressor in human lung adenocarcinoma.

Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics.

Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http://proteomecentral.proteomexchange.org/dataset/PXD001101).