ABSTRACT
PURPOSE: PARP inhibitor resistance may be overcome by combinatorial strategies with agents that disrupt homologous recombination repair (HRR). Multiple HRR pathway components are HSP90 clients, so that HSP90 inhibition leads to abrogation of HRR and sensitisation to PARP inhibition. We performed in vivo preclinical studies of the HSP90 inhibitor onalespib with olaparib and conducted a Phase 1 combination study. PATIENTS AND METHODS: Tolerability and efficacy studies were performed in patient-derived xenograft(PDX) models of ovarian cancer. Clinical safety, tolerability, steady-state pharmacokinetics and preliminary efficacy of olaparib and onalespib were evaluated using a standard 3 + 3 dose-escalation design. RESULTS: Olaparib/onalespib exhibited anti-tumour activity against BRCA1-mutated PDX models with acquired PARPi resistance and PDX models with RB-pathway alterations(CDKN2A loss and CCNE1 overexpression). Phase 1 evaluation revealed that dose levels up to olaparib 300 mg/onalespib 40 mg and olaparib 200 mg/onalespib 80 mg were safe without dose-limiting toxicities. Coadministration of olaparib and onalespib did not appear to affect the steady-state pharmacokinetics of either agent. There were no objective responses, but disease stabilisation ≥24 weeks was observed in 7/22 (32%) evaluable patients including patients with BRCA-mutated ovarian cancers and acquired PARPi resistance and patients with tumours harbouring RB-pathway alterations. CONCLUSIONS: Combining onalespib and olaparib was feasible and demonstrated preliminary evidence of anti-tumour activity.
Subject(s)
Antineoplastic Agents , Ovarian Neoplasms , Antineoplastic Agents/therapeutic use , Carcinoma, Ovarian Epithelial , HSP90 Heat-Shock Proteins , Humans , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Phthalazines/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic useABSTRACT
The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Benzeneacetamides/pharmacology , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Mutant Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Thiazoles/pharmacology , Allosteric Regulation/drug effects , Allosteric Site/drug effects , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cetuximab/pharmacology , Disease Models, Animal , Drug Resistance, Multiple/drug effects , Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm/drug effects , Drug Synergism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Mice , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Conformation/drug effects , Protein Multimerization/drug effectsABSTRACT
BACKGROUND: Based on preclinical work, we found that combination of poly (ADP-ribose) polymerase (PARP) inhibitors with drugs that inhibit the homologous recombination repair (HRR) pathway (such as PI3K inhibitors) might sensitise HRR-proficient epithelial ovarian cancers to PARP inhibitors. We aimed to assess the safety and identify the recommended phase 2 dose of the PARP inhibitor olaparib in combination with the PI3K inhibitor alpelisib in patients with epithelial ovarian cancer and in patients with breast cancer. METHODS: In this multicentre, open-label, phase 1b trial following a 3â+â3 dose-escalation design, we recruited patients aged 18 years or older with the following key eligibility criteria: confirmed diagnosis of either recurrent ovarian, fallopian tube, or primary peritoneal cancer of high-grade serous histology; confirmed diagnosis of either recurrent ovarian, fallopian tube, or primary peritoneal cancer of any histology with known germline BRCA mutations; confirmed diagnosis of recurrent breast cancer of triple-negative histology; or confirmed diagnosis of recurrent breast cancer of any histology with known germline BRCA mutations. Additional patients with epithelial ovarian cancer were enrolled in a dose-expansion cohort. Four dose levels were planned: the starting dose level of alpelisib 250 mg once a day plus olaparib 100 mg twice a day (dose level 0); alpelisib 250 mg once a day plus olaparib 200 mg twice a day (dose level 1); alpelisib 300 mg once a day plus olaparib 200 mg twice a day (dose level 2); and alpelisib 200 mg once a day plus olaparib 200 mg twice a day (dose level 3). Both drugs were administered orally, in tablet formulation. The primary objective was to identify the maximum tolerated dose and the recommended phase 2 dose of the combination of alpelisib and olaparib for patients with epithelial ovarian cancer and patients with breast cancer. Analyses included all patients who received at least one dose of the study drugs. The trial is active, but closed to enrolment; follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov, number NCT01623349. FINDINGS: Between Oct 3, 2014, and Dec 21, 2016, we enrolled 34 patients (28 in the dose-escalation cohort and six in the dose-expansion cohort); two in the dose-escalation cohort were ineligible at the day of scheduled study initiation. Maximum tolerated dose and recommended phase 2 dose were identified as alpelisib 200 mg once a day plus olaparib 200 mg twice a day (dose level 3). Considering all dose levels, the most common treatment-related grade 3-4 adverse events were hyperglycaemia (five [16%] of 32 patients), nausea (three [9%]), and increased alanine aminotransferase concentrations (three [9%]). No treatment-related deaths occurred. Dose-limiting toxic effects included hyperglycaemia and fever with decreased neutrophil count. Of the 28 patients with epithelial ovarian cancer, ten (36%) achieved a partial response and 14 (50%) had stable disease according to Response Evaluation Criteria in Solid Tumors 1.1. INTERPRETATION: Combining alpelisib and olaparib is feasible with no unexpected toxic effects. The observed activity provides preliminary clinical evidence of synergism between olaparib and alpelisib, particularly in epithelial ovarian cancer, and warrants further investigation. FUNDING: Ovarian Cancer Dream Team (Stand Up To Cancer, Ovarian Cancer Research Alliance, National Ovarian Cancer Coalition), Breast Cancer Research Foundation, Novartis.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Ovarian Epithelial/drug therapy , Ovarian Neoplasms/drug therapy , Phosphoinositide-3 Kinase Inhibitors/therapeutic use , Phthalazines/therapeutic use , Piperazines/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Thiazoles/therapeutic use , Aged , Carcinoma, Ovarian Epithelial/genetics , Carcinoma, Ovarian Epithelial/pathology , Dose-Response Relationship, Drug , Drug-Related Side Effects and Adverse Reactions , Female , Genome, Human/genetics , Humans , Maximum Tolerated Dose , Middle Aged , Mutation , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Treatment OutcomeABSTRACT
Upregulation of Pim kinases is observed in several types of leukemias and lymphomas. Pim-1, -2, and -3 promote cell proliferation and survival downstream of cytokine and growth factor signaling pathways. AZD1208 is a potent, highly selective, and orally available Pim kinase inhibitor that effectively inhibits all three isoforms at <5 nM or <150 nM in enzyme and cell assays, respectively. AZD1208 inhibited the growth of 5 of 14 acute myeloid leukemia (AML) cell lines tested, and sensitivity correlates with Pim-1 expression and STAT5 activation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells, accompanied by a dose-dependent reduction in phosphorylation of Bcl-2 antagonist of cell death, 4EBP1, p70S6K, and S6, as well as increases in cleaved caspase 3 and p27. Inhibition of p4EBP1 and p-p70S6K and suppression of translation are the most representative effects of Pim inhibition in sensitive AML cell lines. AZD1208 inhibits the growth of MOLM-16 and KG-1a xenograft tumors in vivo with a clear pharmacodynamic-pharmacokinetic relationship. AZD1208 also potently inhibits colony growth and Pim signaling substrates in primary AML cells from bone marrow that are Flt3 wild-type or Flt3 internal tandem duplication mutant. These results underscore the therapeutic potential of Pim kinase inhibition for the treatment of AML.
Subject(s)
Apoptosis/drug effects , Biphenyl Compounds/pharmacology , Cell Proliferation/drug effects , Leukemia, Myeloid, Acute/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Thiazolidines/pharmacology , Animals , Biphenyl Compounds/pharmacokinetics , Blotting, Western , Cell Cycle , Enzyme-Linked Immunosorbent Assay , Female , Humans , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/pathology , Mice , Mice, SCID , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins c-pim-1/metabolism , Thiazolidines/pharmacokinetics , Tissue Distribution , Tumor Cells, CulturedABSTRACT
TGF-ß-activated kinase 1 (TAK1), a member of the MAPK kinase family, plays a key role in B-cell growth and development. In the present study, we examined the potential role of TAK1 as a therapeutic target for lymphoma. Here, we show that the active phosphorylated form of TAK1 is abundantly expressed in a panel of lymphoma cell lines, including mantle cell, anaplastic large cell, and Hodgkin lymphoma cell lines. Silencing TAK1 expression via the use of siRNA inhibited the activation of NF-κB and p38 and induced apoptosis in lymphoma cell lines. Moreover, submicromolar concentrations of AZ-TAK1, a novel ATP-competitive small molecule inhibitor of TAK1, dephosphorylated TAK1, p38, and IκB-α in lymphoma cell lines. These molecular events were associated with the release of cytochrome c into the cytosol, down-regulation of X-linked inhibitor of apoptosis, activation of caspase 9, and induction of apoptosis. We also demonstrate that primary lymphoma cells express TAK1 and pTAK1 and were sensitive to AZ-TAK1-mediated cell death. Collectively, our data demonstrate an essential role for TAK1 in regulating critical survival mechanisms in lymphoma and suggest that it may serve as a therapeutic target.
Subject(s)
Lymphoma, Mantle-Cell/enzymology , MAP Kinase Kinase Kinases/physiology , Apoptosis , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/physiology , Hodgkin Disease/enzymology , Hodgkin Disease/genetics , Hodgkin Disease/pathology , Humans , I-kappa B Proteins/metabolism , Lymphoma, Large-Cell, Anaplastic/enzymology , Lymphoma, Large-Cell, Anaplastic/genetics , Lymphoma, Large-Cell, Anaplastic/pathology , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/pathology , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/chemistry , MAP Kinase Kinase Kinases/genetics , Models, Molecular , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Phosphorylation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , RNA, Small Interfering/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Despite the success of programmed cell death-1 (PD-1) and PD-1 ligand (PD-L1) inhibitors in treating solid tumors, only a proportion of patients respond. Here, we describe a first-in-class bifunctional therapeutic molecule, STAR0602, that comprises an antibody targeting germline Vß6 and Vß10 T cell receptors (TCRs) fused to human interleukin-2 (IL-2) and simultaneously engages a nonclonal mode of TCR activation with costimulation to promote activation and expansion of αß T cell subsets expressing distinct variable ß (Vß) TCR chains. In solution, STAR0602 binds IL-2 receptors in cis with Vß6/Vß10 TCRs on the same T cell, promoting expansion of human Vß6 and Vß10 CD4+ and CD8+ T cells that acquire an atypical central memory phenotype. Monotherapy with a mouse surrogate molecule induced durable tumor regression across six murine solid tumor models, including several refractory to anti-PD-1. Analysis of murine tumor-infiltrating lymphocyte (TIL) transcriptomes revealed that expanded Vß T cells acquired a distinct effector memory phenotype with suppression of genes associated with T cell exhaustion and TCR signaling repression. Sequencing of TIL TCRs also revealed an increased T cell repertoire diversity within targeted Vß T cell subsets, suggesting clonal revival of tumor T cell responses. These immunological and antitumor effects in mice were recapitulated in studies of STAR0602 in nonhuman primates and human ex vivo models, wherein STAR0602 boosted human antigen-specific T cell responses and killing of tumor organoids. Thus, STAR0602 represents a distinct class of T cell-activating molecules with the potential to deliver enhanced antitumor activity in checkpoint inhibitor-refractory settings.
Subject(s)
Neoplasms , Receptors, Antigen, T-Cell, alpha-beta , Humans , Animals , Mice , Receptors, Antigen, T-Cell, alpha-beta/metabolism , CD8-Positive T-Lymphocytes , Programmed Cell Death 1 Receptor/metabolism , Receptors, Antigen, T-Cell/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Antibodies/pharmacologyABSTRACT
Small-cell lung cancer (SCLC) occurs infrequently in never/former light smokers. We sought to study this rare clinical subset through next-generation sequencing (NGS) and by characterizing a representative patient-derived model. We performed targeted NGS, as well as comprehensive pathological evaluation, in 11 never/former light smokers with clinically diagnosed SCLC. We established a patient-derived model from one such patient (DFCI168) harboring an NRASQ61K mutation and characterized the sensitivity of this model to MEK and TORC1/2 inhibitors. Despite the clinical diagnosis of SCLC, the majority (8/11) of cases were either of nonpulmonary origin or of mixed histology and included atypical carcinoid (n = 1), mixed non-small-cell lung carcinoma and SCLC (n = 4), unspecified poorly differentiated carcinoma (n = 1), or small-cell carcinoma from different origins (n = 2). RB1 and TP53 mutations were found in four and five cases, respectively. Predicted driver mutations were detected in EGFR (n = 2), NRAS (n = 1), KRAS (n = 1), BRCA1 (n = 1), and ATM (n = 1), and one case harbored a TMPRSS2-ERG fusion. DFCI168 (NRASQ61K ) exhibited marked sensitivity to MEK inhibitors in vitro and in vivo. The combination of MEK and mTORC1/2 inhibitors synergized to prevent compensatory mTOR activation, resulting in prolonged growth inhibition in this model and in three other NRAS mutant lung cancer cell lines. SCLC in never/former light smokers is rare and is potentially a distinct disease entity comprised of oncogenic driver mutation-harboring carcinomas morphologically and/or clinically mimicking SCLC. Comprehensive pathologic review integrated with genomic profiling is critical in refining the diagnosis and in identifying potential therapeutic options.
Subject(s)
Genetic Heterogeneity , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Molecular Targeted Therapy , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology , Smokers , Aged , Animals , Base Sequence , Cell Line, Tumor , Cell Survival/drug effects , Drug Synergism , Female , GTP Phosphohydrolases/genetics , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/drug therapy , Male , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , Membrane Proteins/genetics , Mice , Middle Aged , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Models, Biological , Mutation/genetics , Neurosecretory Systems/drug effects , Neurosecretory Systems/pathology , Phenotype , Protein Kinase Inhibitors/pharmacology , Signal Transduction , Small Cell Lung Carcinoma/diagnosis , Small Cell Lung Carcinoma/drug therapyABSTRACT
The clinical efficacy of epidermal growth factor receptor (EGFR)targeted therapy in EGFR-mutant nonsmall cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitorresistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.
Subject(s)
ErbB Receptors , Lung Neoplasms , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic useABSTRACT
BACKGROUND: Tumor orchestrated metabolic changes in the microenvironment limit generation of anti-tumor immune responses. Availability of arginine, a semi-essential amino acid, is critical for lymphocyte proliferation and function. Levels of arginine are regulated by the enzymes arginase 1,2 and nitric oxide synthase (NOS). However, the role of arginase activity in lung tumor maintenance has not been investigated in clinically relevant orthotopic tumor models. METHODS: RNA sequencing (RNA-seq) of sorted cell populations from mouse lung adenocarcinomas derived from immunocompetent genetically engineered mouse models (GEMM)s was performed. To complement mouse studies, a patient tissue microarray consisting of 150 lung adenocarcinomas, 103 squamous tumors, and 54 matched normal tissue were stained for arginase, CD3, and CD66b by multiplex immunohistochemistry. Efficacy of a novel arginase inhibitor compound 9 in reversing arginase mediated T cell suppression was determined in splenocyte ex vivo assays. Additionally, the anti-tumor activity of this compound was determined in vitro and in an autochthonous immunocompetent KrasG12D GEMM of lung adenocarcinoma model. RESULTS: Analysis of RNA-seq of sorted myeloid cells suggested that arginase expression is elevated in myeloid cells in the tumor as compared to the normal lung tissue. Accordingly, in the patient samples arginase 1 expression was mainly localized in the granulocytic myeloid cells and significantly elevated in both lung adenocarcinoma and squamous tumors as compared to the controls. Our ex vivo analysis demonstrated that myeloid derived suppressor cell (MDSC)s cause T cell suppression by arginine depletion, and suppression of arginase activity by a novel ARG1/2 inhibitor, compound 9, led to restoration of T cell function by increasing arginine. Treatment of KrasG12D GEMM of lung cancer model with compound 9 led to a significant tumor regression associated with increased T cell numbers and function, while it had no activity across several murine and human non-small cell (NSCLC) lung cancer lines in vitro. CONCLUSIONS: We show that arginase expression is elevated in mouse and patient lung tumors. In a KRASG12D GEMM arginase inhibition diminished growth of established tumors. Our data suggest arginase as an immunomodulatory target that should further be investigated in lung tumors with high arginase activity.
Subject(s)
Arginase/antagonists & inhibitors , Carcinoma, Non-Small-Cell Lung/enzymology , Lung Neoplasms/enzymology , Myeloid Cells/enzymology , Adult , Aged , Aged, 80 and over , Animals , Arginase/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/immunology , Cell Line, Tumor , Disease Models, Animal , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Mice , Middle Aged , RNA-SeqABSTRACT
Most patients with late-stage high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy but inevitably relapse and develop resistance, highlighting the need for novel therapies to improve patient outcomes. The MEK/ERK pathway is activated in a large subset of HGSOC, making it an attractive therapeutic target. Here, we systematically evaluated the extent of MEK/ERK pathway activation and efficacy of pathway inhibition in a large panel of well-annotated HGSOC patient-derived xenograft models. The vast majority of models were nonresponsive to the MEK inhibitor cobimetinib (GDC-0973) despite effective pathway inhibition. Proteomic analyses of adaptive responses to GDC-0973 revealed that GDC-0973 upregulated the proapoptotic protein BIM, thus priming the cells for apoptosis regulated by BCL2-family proteins. Indeed, combination of both MEK inhibitor and dual BCL-2/XL inhibitor (ABT-263) significantly reduced cell number, increased cell death, and displayed synergy in vitro in most models. In vivo, GDC-0973 and ABT-263 combination was well tolerated and resulted in greater tumor growth inhibition than single agents. Detailed proteomic and correlation analyses identified two subsets of responsive models-those with high BIM at baseline that was increased with MEK inhibition and those with low basal BIM and high pERK levels. Models with low BIM and low pERK were nonresponsive. Our findings demonstrate that combined MEK and BCL-2/XL inhibition has therapeutic activity in HGSOC models and provide a mechanistic rationale for the clinical evaluation of this drug combination as well as the assessment of the extent to which BIM and/or pERK levels predict drug combination effectiveness in chemoresistant HGSOC.
Subject(s)
Cystadenocarcinoma, Serous/drug therapy , Ovarian Neoplasms/drug therapy , Proto-Oncogene Proteins c-bcl-2/genetics , bcl-X Protein/genetics , Aniline Compounds/pharmacology , Animals , Apoptosis/drug effects , Bcl-2-Like Protein 11/genetics , Cell Line, Tumor , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/pathology , Female , Gene Expression Regulation, Neoplastic/drug effects , Heterografts , Humans , MAP Kinase Signaling System/drug effects , Mice , Neoplasm Grading , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Proteomics/methods , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfonamides/pharmacology , bcl-X Protein/antagonists & inhibitors , eIF-2 Kinase/geneticsABSTRACT
PURPOSE: PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance. EXPERIMENTAL DESIGN: Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed. RESULTS: Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability. CONCLUSIONS: Prexasertib exhibits monotherapy activity in PARP inhibitor-resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Checkpoint Kinase 1/antagonists & inhibitors , Cystadenocarcinoma, Serous/drug therapy , Ovarian Neoplasms/drug therapy , Pyrazines/pharmacology , Pyrazoles/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , BRCA1 Protein/genetics , Cell Line, Tumor , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/pathology , DNA Damage/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Female , Humans , Neoplasm Grading , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Phthalazines/pharmacology , Phthalazines/therapeutic use , Piperazines/pharmacology , Piperazines/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyrazines/therapeutic use , Pyrazoles/therapeutic use , Recombinational DNA Repair/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti-PD-1 alone was ineffective, the erdafitinib and anti-PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti-PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti-PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Immunity/drug effects , Neoplasms/immunology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Receptors, Fibroblast Growth Factor/antagonists & inhibitors , Animals , Biomarkers , Cell Line, Tumor , Disease Models, Animal , Drug Synergism , Humans , Immunophenotyping , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Mice , Mice, Transgenic , Mutation , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Prognosis , Programmed Cell Death 1 Receptor/genetics , Pyrazoles/pharmacology , Quinoxalines/pharmacology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, Fibroblast Growth Factor/genetics , Receptors, Fibroblast Growth Factor/metabolism , Signal Transduction/drug effects , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Treatment Outcome , Tumor MicroenvironmentABSTRACT
High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.
Subject(s)
Antineoplastic Agents/metabolism , CDC2 Protein Kinase/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Ovarian Neoplasms/pathology , Phenylenediamines/metabolism , Proto-Oncogene Proteins c-myc/biosynthesis , Pyrimidines/metabolism , Animals , Antineoplastic Agents/administration & dosage , Cell Line, Tumor , Disease Models, Animal , Down-Regulation , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/metabolism , Female , Heterografts , Humans , Mice, SCID , Neoplasm Transplantation , Ovarian Neoplasms/drug therapy , Phenylenediamines/administration & dosage , Pyrimidines/administration & dosage , Treatment Outcome , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
PURPOSE: MET inhibitors can be effective therapies in patients with MET exon 14 (METex14) mutant non-small cell lung cancer (NSCLC). However, long-term efficacy is limited by the development of drug resistance. In this study, we characterize acquired amplification of wild-type (WT) KRAS as a molecular mechanism behind crizotinib resistance in three cases of METex14-mutant NSCLC and propose a combination therapy to target it. EXPERIMENTAL DESIGN: The patient-derived cell line and xenograft (PDX) DFCI358 were established from a crizotinib-resistant METex14-mutant patient tumor with massive focal amplification of WT KRAS. To characterize the mechanism of KRAS-mediated resistance, molecular signaling was analyzed in the parental cell line and its KRAS siRNA-transfected derivative. Sensitivity of the cell line to ligand stimulation was assessed and KRAS-dependent expression of EGFR ligands was quantified. Drug combinations were screened for efficacy in vivo and in vitro using viability and apoptotic assays. RESULTS: KRAS amplification is a recurrent genetic event in crizotinib-resistant METex14-mutant NSCLC. The key characteristics of this genetic signature include uncoupling MET from downstream effectors, relative insensitivity to dual MET/MEK inhibition due to compensatory induction of PI3K signaling, KRAS-induced expression of EGFR ligands and hypersensitivity to ligand-dependent and independent activation, and reliance on PI3K signaling upon MET inhibition. CONCLUSIONS: Using patient-derived cell line and xenografts, we characterize the mechanism of crizotinib resistance mediated by KRAS amplification in METex14-mutant NSCLC and demonstrate the superior efficacy of the dual MET/PI3K inhibition as a therapeutic strategy addressing this resistance mechanism.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Drug Resistance, Neoplasm/genetics , Exons , Gene Amplification , Lung Neoplasms/genetics , Mutation , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Crizotinib/pharmacology , DNA Copy Number Variations , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Humans , In Situ Hybridization, Fluorescence , Lung Neoplasms/diagnosis , Lung Neoplasms/drug therapy , Mice , Models, Biological , Phosphatidylinositol 3-Kinases/genetics , Positron Emission Tomography Computed Tomography , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Animals , Cell Culture Techniques , Cell Line, Tumor , Cytokines/metabolism , Drug Resistance, Neoplasm , Flow Cytometry , Humans , Immunohistochemistry , Immunophenotyping , Mice , Microfluidic Analytical Techniques , Programmed Cell Death 1 Receptor/metabolism , Spheroids, Cellular , Time-Lapse Imaging , Tumor Cells, CulturedABSTRACT
Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies.Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Jenkins et al., p. 196This article is highlighted in the In This Issue feature, p. 127.
Subject(s)
Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Neoplasms/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Immunological/pharmacology , Cell Line, Tumor , Humans , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Mice , Neoplasms/diagnosis , Neoplasms/genetics , Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , T-Lymphocytes/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The lack of effective chemotherapies for high-grade serous ovarian cancers (HGS-OvCa) has motivated a search for alternative treatment strategies. Here, we present an unbiased systems-approach to interrogate a panel of 14 well-annotated HGS-OvCa patient-derived xenografts for sensitivity to PI3K and PI3K/mTOR inhibitors and uncover cell death vulnerabilities. Proteomic analysis reveals that PI3K/mTOR inhibition in HGS-OvCa patient-derived xenografts induces both pro-apoptotic and anti-apoptotic signaling responses that limit cell killing, but also primes cells for inhibitors of anti-apoptotic proteins. In-depth quantitative analysis of BCL-2 family proteins and other apoptotic regulators, together with computational modeling and selective anti-apoptotic protein inhibitors, uncovers new mechanistic details about apoptotic regulators that are predictive of drug sensitivity (BIM, caspase-3, BCL-XL) and resistance (MCL-1, XIAP). Our systems-approach presents a strategy for systematic analysis of the mechanisms that limit effective tumor cell killing and the identification of apoptotic vulnerabilities to overcome drug resistance in ovarian and other cancers.High-grade serous ovarian cancers (HGS-OvCa) frequently develop chemotherapy resistance. Here, the authors through a systematic analysis of proteomic and drug response data of 14 HGS-OvCa PDXs demonstrate that targeting apoptosis regulators can improve response of these tumors to inhibitors of the PI3K/mTOR pathway.
Subject(s)
Antineoplastic Agents/therapeutic use , Apoptosis , Ovarian Neoplasms/pathology , Female , Humans , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Ovarian Neoplasms/drug therapy , Phosphatidylinositol 3-Kinases/metabolism , Systems Biology , TOR Serine-Threonine Kinases/metabolism , X-Linked Inhibitor of Apoptosis Protein/antagonists & inhibitorsABSTRACT
Purpose: Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States, with high rates of recurrence and eventual resistance to cytotoxic chemotherapy. Model systems that allow for accurate and reproducible target discovery and validation are needed to support further drug development in this disease.Experimental Design: Clinically annotated patient-derived xenograft (PDX) models were generated from tumor cells isolated from the ascites or pleural fluid of patients undergoing clinical procedures. Models were characterized by IHC and by molecular analyses. Each PDX was luciferized to allow for reproducible in vivo assessment of intraperitoneal tumor burden by bioluminescence imaging (BLI). Plasma assays for CA125 and human LINE-1 were developed as secondary tests of in vivo disease burden.Results: Fourteen clinically annotated and molecularly characterized luciferized ovarian PDX models were generated. Luciferized PDX models retain fidelity to both the nonluciferized PDX and the original patient tumor, as demonstrated by IHC, array CGH, and targeted and whole-exome sequencing analyses. Models demonstrated diversity in specific genetic alterations and activation of PI3K signaling pathway members. Response of luciferized PDX models to standard-of-care therapy could be reproducibly monitored by BLI or plasma markers.Conclusions: We describe the establishment of a collection of 14 clinically annotated and molecularly characterized luciferized ovarian PDX models in which orthotopic tumor burden in the intraperitoneal space can be followed by standard and reproducible methods. This collection is well suited as a platform for proof-of-concept efficacy and biomarker studies and for validation of novel therapeutic strategies in ovarian cancer. Clin Cancer Res; 23(5); 1263-73. ©2016 AACR.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasms, Glandular and Epithelial/drug therapy , Neoplasms, Glandular and Epithelial/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Xenograft Model Antitumor Assays/methods , Animals , Ascites/pathology , CA-125 Antigen/blood , Carcinoma, Ovarian Epithelial , Cell Line, Tumor , Disease Models, Animal , Humans , Long Interspersed Nucleotide Elements/genetics , Membrane Proteins/blood , Mice , Neoplasms, Glandular and Epithelial/blood , Neoplasms, Glandular and Epithelial/pathology , Ovarian Neoplasms/blood , Ovarian Neoplasms/pathology , Signal Transduction/drug effectsABSTRACT
This corrects the article DOI: 10.1038/ncomms14922.
ABSTRACT
Adenosquamous lung tumours, which are extremely poor prognosis, may result from cellular plasticity. Here, we demonstrate lineage switching of KRAS+ lung adenocarcinomas (ADC) to squamous cell carcinoma (SCC) through deletion of Lkb1 (Stk11) in autochthonous and transplant models. Chromatin analysis reveals loss of H3K27me3 and gain of H3K27ac and H3K4me3 at squamous lineage genes, including Sox2, ΔNp63 and Ngfr. SCC lesions have higher levels of the H3K27 methyltransferase EZH2 than the ADC lesions, but there is a clear lack of the essential Polycomb Repressive Complex 2 (PRC2) subunit EED in the SCC lesions. The pattern of high EZH2, but low H3K27me3 mark, is also prevalent in human lung SCC and SCC regions within ADSCC tumours. Using FACS-isolated populations, we demonstrate that bronchioalveolar stem cells and club cells are the likely cells-of-origin for SCC transitioned tumours. These findings shed light on the epigenetics and cellular origins of lineage-specific lung tumours.