ABSTRACT
PURPOSE: Desmoplastic small round cell tumor (DSRCT) is a rare but highly aggressive soft tissue sarcoma that arises in the abdominopelvic cavity of young males. Since the discovery of EWSR1::WT1 fusion as the driver of DSRCT, no actionable genomic alterations have been identified, limiting disease management to a combination of surgery, chemotherapy, and radiation with very poor outcomes. Herein, we leveraged ERBB2/HER2 expression in DSRCT as a therapeutic target. EXPERIMENTAL DESIGN: ERBB2/HER2 expression was evaluated in clinical samples and patient-derived xenografts (PDX) using RNA-seq, RT-qPCR, and a newly developed HER2 IHC assay (Clone 29D8). Responses to HER2 antibody-drug conjugates (ADC) -trastuzumab deruxtecan (DS-8201, T-DXd) and trastuzumab emtansine (T-DM1)- were evaluated in DSRCT-PDX, cell line, and organoid models. Drug internalization was demonstrated by live microscopy. Apoptosis was evaluated by Western blotting and caspase activity assays. RESULTS: ERBB2/HER2 was detectable in DSRCT samples from patients and PDXs, with higher sensitivity of RNA assays and improved IHC detectability using Clone 29D8. Treatment of ERBB2/HER2-expressing DSRCT PDX, cell line, and organoid models with T-DXd or T-DM1 resulted in tumor regression. This therapeutic response was long-lasting in T-DXd-treated xenografts and was mediated by rapid HER2-ADC complex internalization and cytotoxicity, triggering p53-mediated apoptosis and growth arrest. Xenograft regression was associated with bystander payload effects triggering global tumor niche responses proportional to HER2 status. Conclusions ERBB2/HER2 is a therapeutic target for DSRCT. HER2-ADCs are novel options for managing this exceptionally aggressive sarcoma and may fulfill its urgent and historically unmet need for more effective clinical therapy.
ABSTRACT
BACKGROUND: Even though BRAF fusions are increasingly detected in standard multigene next-generation sequencing panels, few reports have explored their structure and impact on clinical course. PATIENTS/METHODS: We collected data from patients with BRAF fusion-positive cancers identified through a genotyping protocol of 97,024 samples. Fusions were characterized and reviewed for oncogenic potential (in-frame status, non-BRAF partner gene, intact BRAF kinase domain). RESULTS: We found 241 BRAF fusion-positive tumors from 212 patients with 82 unique 5' fusion partners spanning 52 histologies. 39 fusion partners were not previously reported, and 61 were identified once. BRAF fusion incidence was enriched in pilocytic astrocytomas, gangliomas, low-grade neuroepithelial tumors, and acinar cell carcinoma of the pancreas. 24 patients spanning multiple histologies were treated with MAPK-directed therapies of which 20 were evaluable for RECIST. Best response was partial response (N=2), stable disease (N=11), and progressive disease (N=7). The median time on therapy was 1 month with MEK plus BRAF inhibitors ([N=11], range 0-18 months) and 8 months for MEK inhibitors ([N=14], range 1-26 months). 9 patients remained on treatment for longer than 6 months [pilocytic astrocytomas (N=6), Erdheim-Chester disease (N=1), extraventricular neurocytoma (N=1), melanoma (N=1)]. Fifteen patients had acquired BRAF fusions. CONCLUSIONS: BRAF fusions are found across histologies and represent an emerging actionable target. BRAF fusions have a diverse set of fusion partners. Durable responses to MAPK therapies were seen, particularly in pilocytic astrocytomas. Acquired BRAF fusions were identified after targeted therapy underscoring the importance of post-progression biopsies to optimize treatment at relapse in these patients.
ABSTRACT
Despite the potential of targeted epigenetic therapies, most cancers do not respond to current epigenetic drugs. The Polycomb repressive complex EZH2 inhibitor tazemetostat was recently approved for the treatment of SMARCB1-deficient epithelioid sarcomas, based on the functional antagonism between PRC2 and loss of SMARCB1. Through the analysis of tazemetostat-treated patient tumors, we recently defined key principles of their response and resistance to EZH2 epigenetic therapy. Here, using transcriptomic inference from SMARCB1-deficient tumor cells, we nominate the DNA damage repair kinase ATR as a target for rational combination EZH2 epigenetic therapy. We show that EZH2 inhibition promotes DNA damage in epithelioid and rhabdoid tumor cells, at least in part via its induction of the transposase-derived PGBD5. We leverage this collateral synthetic lethal dependency to target PGBD5-dependent DNA damage by inhibition of ATR but not CHK1 using elimusertib. Consequently, combined EZH2 and ATR inhibition improves therapeutic responses in diverse patient-derived epithelioid and rhabdoid tumors in vivo. This advances a combination epigenetic therapy based on EZH2-PGBD5 synthetic lethal dependency suitable for immediate translation to clinical trials for patients.
ABSTRACT
Epigenetic dependencies have become evident in many cancers. On the basis of antagonism between BAF/SWI-SNF and PRC2 in SMARCB1-deficient sarcomas, we recently completed the clinical trial of the EZH2 inhibitor tazemetostat. However, the principles of tumor response to epigenetic therapy in general, and tazemetostat in particular, remain unknown. Using functional genomics and diverse experimental models, we define molecular mechanisms of tazemetostat resistance in SMARCB1-deficient tumors. We found distinct acquired mutations that converge on the RB1/E2F axis and decouple EZH2-dependent differentiation and cell-cycle control. This allows tumor cells to escape tazemetostat-induced G1 arrest, suggests a general mechanism for effective therapy, and provides prospective biomarkers for therapy stratification, including PRICKLE1. On the basis of this, we develop a combination strategy to circumvent tazemetostat resistance using bypass targeting of AURKB. This offers a paradigm for rational epigenetic combination therapy suitable for translation to clinical trials for epithelioid sarcomas, rhabdoid tumors, and other epigenetically dysregulated cancers. SIGNIFICANCE: Genomic studies of patient epithelioid sarcomas and rhabdoid tumors identify mutations converging on a common pathway for response to EZH2 inhibition. Resistance mutations decouple drug-induced differentiation from cell-cycle control. We identify an epigenetic combination strategy to overcome resistance and improve durability of response, supporting its investigation in clinical trials. See related commentary by Paolini and Souroullas, p. 903. This article is featured in Selected Articles from This Issue, p. 897.
Subject(s)
Drug Resistance, Neoplasm , Enhancer of Zeste Homolog 2 Protein , Epigenesis, Genetic , Pyridones , Humans , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Enhancer of Zeste Homolog 2 Protein/genetics , Pyridones/therapeutic use , Pyridones/pharmacology , Drug Resistance, Neoplasm/genetics , Morpholines/pharmacology , Morpholines/therapeutic use , Animals , Mice , Biphenyl Compounds/therapeutic use , Biphenyl Compounds/pharmacology , Cell Line, Tumor , SMARCB1 Protein/genetics , Benzamides/therapeutic use , Benzamides/pharmacology , MutationABSTRACT
INTRODUCTION: NRG1 gene fusions are clinically actionable alterations identified in NSCLC and other tumors. Previous studies have reported that NRG1 fusions signal through HER2 and HER3 but, thus far, strategies targeting HER3 specifically or HER2-HER3 signaling have exhibited modest activity in patients with NSCLC bearing NRG1 fusions. Although NRG1 fusion proteins can bind HER4 in addition to HER3, the contribution of HER4 and other HER family members in NRG1 fusion-positive cancers is not fully understood. METHODS: We investigated the role of HER4 and EGFR-HER3 signaling in NRG1 fusion-positive cancers using Ba/F3 models engineered to express various HER family members in combination with NRG1 fusions and in vitro and in vivo models of NRG1 fusion-positive cancer. RESULTS: We determined that NRG1 fusions can stimulate downstream signaling and tumor cell growth through HER4, independent of other HER family members. Moreover, EGFR-HER3 signaling is also activated in cells expressing NRG1 fusions, and inhibition of these receptors is also necessary to effectively inhibit tumor cell growth. We observed that cetuximab, an anti-EGFR antibody, in combination with anti-HER2 antibodies, trastuzumab and pertuzumab, yielded a synergistic effect. Furthermore, pan-HER tyrosine kinase inhibitors were more effective than tyrosine kinase inhibitors with greater specificity for EGFR, EGFR-HER2, or HER2-HER4, although the relative degree of dependence on EGFR or HER4 signaling varied between different NRG1 fusion-positive cancers. CONCLUSIONS: Our findings indicate that pan-HER inhibition including HER4 and EGFR blockade is more effective than selectively targeting HER3 or HER2-HER3 in NRG1 fusion-positive cancers.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , ErbB Receptors/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Neuregulin-1/genetics , Neuregulin-1/metabolism , Receptor, ErbB-2 , Receptor, ErbB-3/genetics , Receptor, ErbB-3/metabolism , Signal TransductionABSTRACT
INTRODUCTION: ERBB2 amplification in lung cancer remains poorly characterized. HER2 (encoded by ERBB2) is a transmembrane tyrosine kinase capable of ligand-independent dimerization and signaling when overexpressed, and a common cause of HER2 overexpression is ERBB2 amplification. Here, we evaluated the clinicopathologic and genomic characteristics of ERBB2-amplified NSCLC and explored a HER2 antibody-drug conjugate (ADC) therapeutic strategy. METHODS: Our institutional next-generation DNA sequencing data (OncoPanel) from 5769 NSCLC samples (5075 patients) were queried for cases having high-level ERBB2 amplification (≥6 copies). Clinical and demographic characteristics were extracted from the electronic medical records. Efficacy of the pan-ERBB inhibitor afatinib or HER2 ADCs (trastuzumab deruxtecan and trastuzumab emtansine) was evaluated in NSCLC preclinical models and patients with ERBB2 amplification. RESULTS: High-level ERBB2 amplification was identified in 0.9% of lung adenocarcinomas and reliably predicted overexpression of HER2. ERBB2 amplification events are detected in two distinct clinicopathologic and genomic subsets of NSCLC: as the sole mitogenic driver in tumors arising in patients with a smoking history or as a concomitant alteration with other mitogenic drivers in patients with a light or never smoking history. We further reveal that trastuzumab deruxtecan is effective therapy in in vitro and in vivo preclinical models of NSCLC harboring ERBB2 amplification and report two cases of clinical activity of an anti-HER2 ADC in patients who acquired ERBB2 amplification after previous targeted therapy. CONCLUSIONS: High-level ERBB2 amplification reliably predicts HER2 overexpression in patients with NSCLC, and HER2 ADC is effective therapy in this population.
Subject(s)
Camptothecin/analogs & derivatives , Carcinoma, Non-Small-Cell Lung , Gene Amplification , Lung Neoplasms , Receptor, ErbB-2 , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Receptor, ErbB-2/metabolism , Receptor, ErbB-2/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Female , Male , Middle Aged , Animals , Immunoconjugates/therapeutic use , Immunoconjugates/pharmacology , Aged , Mice , Trastuzumab/therapeutic use , Trastuzumab/pharmacology , Prevalence , Afatinib/therapeutic use , Afatinib/pharmacology , Ado-Trastuzumab Emtansine/therapeutic use , Ado-Trastuzumab Emtansine/pharmacologyABSTRACT
RET receptor tyrosine kinase is activated in various cancers (lung, thyroid, colon and pancreatic, among others) through oncogenic fusions or gain-of-function single-nucleotide variants. Small-molecule RET kinase inhibitors became standard-of-care therapy for advanced malignancies driven by RET. The therapeutic benefit of RET inhibitors is limited, however, by acquired mutations in the drug target as well as brain metastasis, presumably due to inadequate brain penetration. Here, we perform preclinical characterization of vepafestinib (TAS0953/HM06), a next-generation RET inhibitor with a unique binding mode. We demonstrate that vepafestinib has best-in-class selectivity against RET, while exerting activity against commonly reported on-target resistance mutations (variants in RETL730, RETV804 and RETG810), and shows superior pharmacokinetic properties in the brain when compared to currently approved RET drugs. We further show that these properties translate into improved tumor control in an intracranial model of RET-driven cancer. Our results underscore the clinical potential of vepafestinib in treating RET-driven cancers.
Subject(s)
Brain Neoplasms , Mutation , Brain , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , SolventsABSTRACT
INTRODUCTION: Although targeted therapies have revolutionized the therapeutic landscape of lung adenocarcinomas (LUADs), disease progression on single-agent targeted therapy against known oncogenic drivers is common, and therapeutic options after disease progression are limited. In patients with MDM2 amplification (MDM2amp) and a concurrent oncogenic driver alteration, we hypothesized that targeting of the tumor-suppressor pathway (by means of restoration of p53 using MDM2 inhibition) and simultaneous targeting of co-occurring MAPK oncogenic pathway might represent a more durably effective therapeutic strategy. METHODS: We evaluated genomic next-generation sequencing data using the Memorial Sloan Kettering Cancer Center-Integrated Mutation Profiling of Actionable Cancer Targets platform to nominate potential targets for combination therapy in LUAD. We investigated the small molecule MDM2 inhibitor milademetan in cell lines and patient-derived xenografts of LUAD with a known driver alteration and MDM2amp. RESULTS: Of 10,587 patient samples from 7121 patients with LUAD profiled by next-generation sequencing, 6% (410 of 7121) harbored MDM2amp. MDM2amp was significantly enriched among tumors with driver alterations in METex14 (36%, p < 0.001), EGFR (8%, p < 0.001), RET (12%, p < 0.01), and ALK (10%, p < 0.01). The combination of milademetan and the MEK inhibitor trametinib was synergistic in growth inhibition of ECLC5-GLx (TRIM33-RET/MDM2amp), LUAD12c (METex14/KRASG12S/MDM2amp), SW1573 (KRASG12C, TP53 wild type), and A549 (KRASG12S) cells and in increasing expression of proapoptotic proteins PUMA and BIM. Treatment of ECLC5-GLx and LUAD12c with single-agent milademetan increased ERK phosphorylation, consistent with previous data on ERK activation with MDM2 inhibition. This ERK activation was effectively suppressed by concomitant administration of trametinib. In contrast, ERK phosphorylation induced by milademetan was not suppressed by concurrent RET inhibition using selpercatinib (in ECLC5-GLx) or MET inhibition using capmatinib (in LUAD12c). In vivo, combination milademetan and trametinib was more effective than either agent alone in ECLC5-GLx, LX-285 (EGFRex19del/MDM2amp), L13BS1 (METex14/MDM2amp), and A549 (KRASG12S, TP53 wild type). CONCLUSIONS: Combined MDM2/MEK inhibition was found to have efficacy across multiple patient-derived LUAD models harboring MDM2amp and concurrent oncogenic drivers. This combination, potentially applicable to LUADs with a wide variety of oncogenic driver mutations and kinase fusions activating the MAPK pathway, has evident clinical implications and will be investigated as part of a planned phase 1/2 clinical trial.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Mutation , Disease Progression , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/therapeutic use , Proto-Oncogene Proteins c-mdm2/genetics , Transcription Factors/geneticsABSTRACT
Essential epigenetic dependencies have become evident in many cancers. Based on the functional antagonism between BAF/SWI/SNF and PRC2 in SMARCB1-deficient sarcomas, we and colleagues recently completed the clinical trial of the EZH2 inhibitor tazemetostat. However, the principles of tumor response to epigenetic therapy in general, and tazemetostat in particular, remain unknown. Using functional genomics of patient tumors and diverse experimental models, we sought to define molecular mechanisms of tazemetostat resistance in SMARCB1-deficient sarcomas and rhabdoid tumors. We found distinct classes of acquired mutations that converge on the RB1/E2F axis and decouple EZH2-dependent differentiation and cell cycle control. This allows tumor cells to escape tazemetostat-induced G1 arrest despite EZH2 inhibition, and suggests a general mechanism for effective EZH2 therapy. This also enables us to develop combination strategies to circumvent tazemetostat resistance using cell cycle bypass targeting via AURKB, and synthetic lethal targeting of PGBD5-dependent DNA damage repair via ATR. This reveals prospective biomarkers for therapy stratification, including PRICKLE1 associated with tazemetostat resistance. In all, this work offers a paradigm for rational epigenetic combination therapy suitable for immediate translation to clinical trials for epithelioid sarcomas, rhabdoid tumors, and other epigenetically dysregulated cancers.
ABSTRACT
Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive pediatric cancer driven by the EWSR1-WT1 fusion oncogene. Combinations of chemotherapy, radiation and surgery are not curative, and the 5-years survival rate is less than 25%. One potential explanation for refractoriness is the existence of a cancer stem cell (CSC) subpopulation able escape current treatment modalities. However, no study to-date has examined the role of CSCs in DSRCT or established in vitro culture conditions to model this subpopulation. In this study, we investigated the role of stemness markers in DSRCT survival and metastasis, finding that elevated levels of SOX2 and NANOG are associated with worse survival in sarcoma patients and are elevated in metastatic DSRCT tumors. We further develop the first in vitro DSRCT CSC model which forms tumorspheres, expresses increased levels of stemness markers (SOX2, NANOG, KLF4, and OCT4), and resists doxorubicin chemotherapy treatment. This model is an important addition to the DSRCT tool kit and will enable investigation of this critical DSRCT subpopulation. Despite lower sensitivity to chemotherapy, the DSRCT CSC model remained sensitive to knockdown of the EWSR1-WT1 fusion protein, suggesting that future therapies directed against this oncogenic driver have the potential to treat both DSRCT bulk tumor and CSCs.
ABSTRACT
MEK inhibitors (MEKi) have limited efficacy in KRAS mutant lung adenocarcinoma (LUAD) patients, and this is attributed to both intrinsic and adaptive mechanisms of drug resistance. While many studies have focused on the former, there remains a dearth of data regarding acquired resistance to MEKi in LUAD. We established trametinib-resistant KRAS mutant LUAD cells through dose escalation and performed targeted MSK-IMPACT sequencing to identify drivers of MEKi resistance. Comparing resistant cells to their sensitive counterparts revealed alteration of genes associated with trametinib response. We describe a state of "drug addiction" in resistant cases where cells are dependent on continuous culture in trametinib for survival. We show that dependence on ERK2 suppression underlies this phenomenon and that trametinib removal hyperactivates ERK, resulting in ER stress and apoptosis. Amplification of KRASG12C occurs in drug-addicted cells and blocking mutant-specific activity with AMG 510 rescues the lethality associated with trametinib withdrawal. Furthermore, we show that increased KRASG12C expression is lethal to other KRAS mutant LUAD cells, consequential to ERK hyperactivation. Our study determines the drug-addicted phenotype in lung cancer is associated with KRAS amplification and demonstrates that toxic acquired genetic changes can develop de novo in the background of MAPK suppression with MEK inhibitors. We suggest that the presence of mutant KRAS amplification in patients may identify those that may benefit from a "drug holiday" to circumvent drug resistance. These findings demonstrate the toxic potential of hyperactive ERK signaling and highlight potential therapeutic opportunities in patients bearing KRAS mutations.
ABSTRACT
Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive malignant cancer caused by a chromosomal translocation t(11;22)(p13;q12) that produces an oncogenic transcription factor, EWSR1-WT1. EWSR1-WT1 is essential for the initiation and progression of DSRCT. However, the precise mechanism by which EWSR1-WT1 drives DSRCT oncogenesis remains unresolved. Through our integrative gene expression analysis, we identified Salt Inducible Kinase 1 (SIK1) as a direct target of EWSR1-WT1. SIK1 as a member of the AMPK related kinase is involved in many biological processes. We showed that depletion of SIK1 causes inhibition of tumor cell growth, similar to the growth inhibition observed when EWSR1-WT1 is depleted. We further showed that silencing SIK1 leads to cessation of DNA replication in DSRCT cells and inhibition of tumor growth in vivo. Lastly, combined inhibition of SIK1 and CHEK1with small molecule inhibitors, YKL-05-099 and prexasertib, respectively, showed enhanced cytotoxicity in DSRCT cells compared to inhibition of either kinases alone. This work identified SIK1 as a new potential therapeutic target in DSRCT and the efficacy of SIK1 inhibition may be improved when combined with other intervention strategies.
ABSTRACT
Activating RAS mutations are found in a subset of fusion-negative rhabdomyosarcoma (RMS), and therapeutic strategies to directly target RAS in these tumors have been investigated, without clinical success to date. A potential strategy to inhibit oncogenic RAS activity is the disruption of RAS prenylation, an obligate step for RAS membrane localization and effector pathway signaling, through inhibition of farnesyltransferase (FTase). Of the major RAS family members, HRAS is uniquely dependent on FTase for prenylation, whereas NRAS and KRAS can utilize geranylgeranyl transferase as a bypass prenylation mechanism. Tumors driven by oncogenic HRAS may therefore be uniquely sensitive to FTase inhibition. To investigate the mutation-specific effects of FTase inhibition in RMS we utilized tipifarnib, a potent and selective FTase inhibitor, in in vitro and in vivo models of RMS genomically characterized for RAS mutation status. Tipifarnib reduced HRAS processing, and plasma membrane localization leading to decreased GTP-bound HRAS and decreased signaling through RAS effector pathways. In HRAS-mutant cell lines, tipifarnib reduced two-dimensional and three-dimensional cell growth, and in vivo treatment with tipifarnib resulted in tumor growth inhibition exclusively in HRAS-mutant RMS xenografts. Our data suggest that small molecule inhibition of FTase is active in HRAS-driven RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with RMS.
Subject(s)
Rhabdomyosarcoma, Embryonal , Rhabdomyosarcoma , Farnesyltranstransferase/genetics , Genes, ras , Humans , Prenylation , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/geneticsABSTRACT
Targeted therapies for MET exon 14-skipping (METΔex14)-driven lung cancers have generated some promising results but response rates remain below that seen for other kinase-driven cancers. One strategy for improving treatment outcomes is to employ rational combination therapies to enhance the suppression of tumour growth and delay or prevent the emergence of resistance. To this end, we profiled the transcriptomes of MET-addicted lung tumours and cell lines and identified the RAS-mitogen-activated protein kinase (MAPK) pathway as a critical effector required for METΔex14-dependent growth. Ectopic expression of MET in an isogenic cell line model showed that overexpression of the mutant MET receptor led to higher levels of MAPK phosphorylation and nuclear import, resulting in increased expression and phosphorylation of nuclear MAPK targets. In comparison, other known MET effectors were unaffected. Inhibition of this pathway by KRAS knockdown in MET-addicted cells in vitro led to decreased viability in only the METΔex14-mutant cells. Conversely, decoupling RAS-MAPK axis, but not other effector pathways, from MET activity via the introduction of constitutively active mutants conferred resistance to MET inhibitors in vitro. Our results suggest that aberrant hyperactivity of the MET receptor caused by the exon 14-skipping mutation does not uniformly upregulate all known downstream effectors, rather gaining a predilection for aberrantly activating and subsequently relying on the RAS-MAPK pathway. These findings provide a rationale for the co-targeting of the RAS-MAPK pathway alongside MET to prolong therapeutic response and circumvent resistance to improve patient survival.
ABSTRACT
Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.
Subject(s)
Lung Neoplasms/pathology , Oxidative Stress/physiology , Oxidoreductases/metabolism , Thioredoxin-Disulfide Reductase/metabolism , Glutathione/metabolism , Humans , Lung/metabolism , Lung/pathology , Lung Neoplasms/metabolism , Reactive Oxygen Species/metabolism , Thioredoxins/metabolismABSTRACT
NRG1 rearrangements are recurrent oncogenic drivers in solid tumors. NRG1 binds to HER3, leading to heterodimerization with other HER/ERBB kinases, increased downstream signaling, and tumorigenesis. Targeting ERBBs, therefore, represents a therapeutic strategy for these cancers. We investigated zenocutuzumab (Zeno; MCLA-128), an antibody-dependent cellular cytotoxicity-enhanced anti-HER2xHER3 bispecific antibody, in NRG1 fusion-positive isogenic and patient-derived cell lines and xenograft models. Zeno inhibited HER3 and AKT phosphorylation, induced expression of apoptosis markers, and inhibited growth. Three patients with chemotherapy-resistant NRG1 fusion-positive metastatic cancer were treated with Zeno. Two patients with ATP1B1-NRG1-positive pancreatic cancer achieved rapid symptomatic, biomarker, and radiographic responses and remained on treatment for over 12 months. A patient with CD74-NRG1-positive non-small cell lung cancer who had progressed on six prior lines of systemic therapy, including afatinib, responded rapidly to treatment with a partial response. Targeting HER2 and HER3 simultaneously with Zeno is a novel therapeutic paradigm for patients with NRG1 fusion-positive cancers. SIGNIFICANCE: NRG1 rearrangements encode chimeric ligands that activate the ERBB receptor tyrosine kinase family. Here we show that targeting HER2 and HER3 simultaneously with the bispecific antibody Zeno leads to durable clinical responses in patients with NRG1 fusion-positive cancers and is thus an effective therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1171.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Antibodies, Bispecific , Carcinogenesis/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Cell Line, Tumor , Gene Rearrangement , Humans , Immunoglobulin G , Lung Neoplasms/genetics , Neuregulin-1/genetics , Receptor, ErbB-2 , Receptor, ErbB-3/genetics , Receptor, ErbB-3/metabolismABSTRACT
Targeted therapy of ROS1-fusion-driven non-small cell lung cancer (NSCLC) has achieved notable clinical success. Despite this, resistance to therapy inevitably poses a significant challenge. MYC amplification was present in â¼19% of lorlatinib-resistant ROS1-driven NSCLC. We hypothesized that MYC overexpression drives ROS1-TKI resistance. Using complementary approaches in multiple models, including a MYC-amplified patient-derived cell line and xenograft (LUAD-0006), we established that MYC overexpression induces broad ROS1-TKI resistance. Pharmacologic inhibition of ROS1 combined with MYC knockdown were essential to completely suppress LUAD-0006 cell proliferation compared with either treatment alone. We interrogated cellular signaling in ROS1-TKI-resistant LUAD-0006 and discovered significant differential regulation of targets associated with cell cycle, apoptosis, and mitochondrial function. Combinatorial treatment of mitochondrial inhibitors with crizotinib revealed inhibitory synergism, suggesting increased reliance on glutamine metabolism and fatty-acid synthesis in chronic ROS1-TKI treated LUAD-0006 cells. In vitro experiments further revealed that CDK4/6 and BET bromodomain inhibitors effectively mitigate ROS1-TKI resistance in MYC-overexpressing cells. Notably, in vivo studies demonstrate that tumor control may be regained by combining ROS1-TKI and CDK4/6 inhibition. Our results contribute to the broader understanding of ROS1-TKI resistance in NSCLC. IMPLICATIONS: This study functionally characterizes MYC overexpression as a novel form of therapeutic resistance to ROS1 tyrosine kinase inhibitors in non-small cell lung cancer and proposes rational combination treatment strategies.