Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for
-mutant non-small cell lung cancers (NSCLC). However, most patients develop acquired ...drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in
-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3-DXd is an antibody-drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3-DXd across a series of EGFR inhibitor-resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3-DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3-DXd. The combination of osimertinib and HER3-DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients. SIGNIFICANCE: EGFR inhibition leads to increased HER3 membrane expression and promotes HER3-DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3-DXd combination in EGFR-mutant lung cancer.
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Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. ...Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit.
modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. SIGNIFICANCE: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC I
subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection.
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Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed ...cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.
MET-targeted therapies are clinically effective in
-amplified and
exon 14 deletion mutant (
ex14) non-small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug ...resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones
The combination of capmatinib/merestinib was evaluated
and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate
and
that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.
Bioluminescence imaging (BLI) is an established method for evaluating metastatic load in preclinical cancer models; however, BLI can produce observational error due to differences in substrate ...concentration and signal depth. In our syngeneic murine model of metastasis (VM-M3), we used a quantitative polymerase chain reaction (qPCR) method of DNA quantification to bypass these limitations. Liver, spleen, and brain from VM/Dk (VM) mice bearing VM-M3 tumor cells were first imaged ex vivo with BLI. qPCR quantification of tumor cell DNA was then performed on DNA extracted from these organs. Linear regression indicated that qPCR data predicted BLI data in solid tissue. Furthermore, the tumor cell detection limit was lower for qPCR analysis than for BLI analysis. In order to validate qPCR for use in detecting blood metastases, qPCR quantification was performed on whole blood collected from mice whose global organ metastatic load (summation of liver, spleen, kidneys, lungs, and brain) was quantified through BLI. Linear regression indicated that qPCR data in blood predicted BLI data in solid tissue. The results demonstrate that qPCR is an accurate and sensitive method of metastatic quantification in syngeneic murine models.
•A qPCR method is described for measuring metastasis in a syngeneic murine model.•The method detects metastatic tumor cells in solid organ tissues and in blood.•The qPCR method is more sensitive than BLI for detecting circulating tumor cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8
T cells specifically recognizing the model antigen in an H-2b-restricted manner. The assay ...was screened with a small-molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8
cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting
that sensitized tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.
Abstract
The clinical successes achieved by different immunotherapies have resulted in a paradigm shift in treatment modalities. Despite these significant advances, not all patients benefit from the ...use of these therapies, creating a need to develop additional approaches to enhance and broaden their clinical application. To identify genes whose products can increase or decrease the sensitivity of tumor cells to the immune system, we used a CTL assay to screen a whole genomic CRISPR library. We co-culture a mouse cell line, ID8, expressing a model antigen (Ova) with transgenic CD8 T cells (OT-I) recognizing this antigen. A set of controls that enhance or decrease CTL activity behaved as expected. Comparison of the CRISPR score identified several hits that increased or decreased the sensitivity of the tumor cells to CTL killing. Subsets of these hits belong to two pathways involved in CTL-mediated killing: the IFN-γ and the TNF-α signaling pathways. We evaluated which of these hits would be amenable to therapeutic modulation, and decided to focus on the kinase TAK1 for confirmation and validation studies. A TAK1 deficient cell line was more sensitive to CTL killing, which was prevented by expression of TAK1, confirming the role of TAK1 in this process. A TAK1 gene carrying an inactivation mutation K63W did not rescue the effects of TAK1 KO, indicating that TAK1 enzymatic activity was necessary. Several pathways mediate CTL killing: Perforin/Granzyme B, IFN-γ, TNF-α, Fas & TRAIL pathways. To determine TAK1 MOA, we studied the effects of a Perforin/Granzyme B inhibitor CMA. CMA inhibited CTL activity in a dose-dependent manner on WT cells, but did not inhibit CTL activity on TAK1 deficient cells, indicating TAK1 effects are independent of this pathway. We then tested the sensitivity of TAK1 KO cells to TNF-α. TAK1 KO cells were more sensitive to TNF-α mediated killing, and similar results were observed with several additional cell lines (MC38, EMT6, KP). TNF-α can activate the JNK, p38, and NF-κB pathways, and the apoptosis extrinsic pathway to regulate cell growth and cell death. Kinetics studies monitoring pathway activity upon TNF-α stimulation showed that TAK1 KO cell lines induced cFLIP degradation before observing PARP cleavage, and that the NF-κB pathway, which has been observed to mediate cFLIP synthesis, was not activated. We proceeded to evaluate the effects of TAK1 deficiency in a mouse syngeneic model. TAK1 deficiency resulted in reduced growth and increased survival in the MC38 in vivo model. In summary, by screening a CRISPR library against a CTL assay, we identified TAK1 as a novel potential target for immunotherapies. TAK1 deficiency enhances CTL killing and results in decreased tumor growth and increased survival in vivo. This results support the development of TAK1 inhibitors to enhance the anti-tumor action of the immune system.
Citation Format: Juan J. Miret, Troy A. Luster, Patrick Lizotte, Min Wu, Sarah Nzikoba, Luke Taus J. Taus, Prafulla C. Gokhale, Paul Kirschmeier, David Barbie, Cloud P. Paweletz. TAK1 deficiency in tumor cells enhances sensitivity to CTL-mediated killing via TNF-α abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5543.
Abstract
Introduction: Immune checkpoint blockade, including αPD-1 and αPD-L1 form the backbone of personalized medicine for lung cancer and other malignancies. Yet the underlying mechanisms of ...resistance to therapy are not fully characterized partly because functional models to perform mechanistic studies are lacking. Here we report on single cell RNA sequencing from murine (or patient) derived organotypic tumor spheroids (DOTS-seq) that enables analysis of tumor and immune cell intrinsic changes that occur during αPD-1 treatment ex vivo.
Methods: Murine-derived organotypic spheroids from syngeneic MC38 tumors were grown and treated with αPD-1 and isotype matched IgG in a microfluidic device as previously described1. At day 6, libraries were prepared from individual viable cells using the 10X Genomics platform and sequenced at DFCI. Sequencing data was processed using the Seurat package and corrected for UMI, ribosomal, mitochondrial and cell cycle transcripts. Dimensionality reduction, clustering, and identification of differentially expressed genes were performed on log normalized data. Gene set enrichment analysis was performed using the SetRank package.
Results: Transcripts were obtained for 2,543 IgG treated and 2,626 αPD-1 treated cells that were resistant to ex vivo killing. 60% of αPD-1 treated cells fell into 2 unique clusters which each had downregulated genes associated with apoptosis and interferon-γ response, such as Dap, Cxcl10 and B2m. Within these two, one cluster contained highly upregulated genes known to be E2F targets or important for G2M transition while the other did not. Interestingly, the quiescent subpopulation exhibited a unique epithelial to mesenchymal transition-like state characterized by expression of Snai1, Mmp2, Mmp14, and Vegfa. This subpopulation also upregulated transcripts of immuno-modulatory cytokines from the IL6 family, including Il11, Lif, and Osm. IL-6 extracellular levels are also elevated in treated cultures, suggesting a mesenchymal-like cell subpopulation responsible for this cytokine modulation of the tumor microenvironment.
Conclusion: Here we show that profiling the interplay between tumor and immune cells at the single cell level is possible ex vivo. We identify a previously uncharacterized subpopulation of EMT-like cells that regulate the tumor microenvironment and promote resistance to αPD-1 in MC38 tumors.
Reference: 1. Jenkins RW, et al. Cancer Discov DOI:10.1158/2159-8290
Citation Format: Andrew J. Portell, Jonathan Greene, Luke J. Taus, Patrick Lizotte, Elena Ivanova, Kalil O. Menezes, Amir R. Aref, Paul Kirschmeier, Russell W. Jenkins, David Barbie, Cloud P. Paweletz. Ex vivo single cell RNA-sequencing of tumor derived organotypic spheroids identifies a unique mesenchymal resistance program to PD-1 blockade abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1483.
Abstract
Genetic alterations which constitutively activate human epidermal growth factor receptor 2 (HER2; ERBB2) without overexpression have been observed in patients with non-small cell lung cancer ...(NSCLC) yet currently there are no effective targeted therapies for such patients. Neratinib is an orally available, quinazoline-based, irreversible pan-HER tyrosine kinase inhibitor. Prior clinical studies have demonstrated that neratinib alone or in combination with temsirolimus has low response rates in patients with HER2 mutated NSCLC. Since dual HER2-tageting is showing promising activity in breast cancer, we hypothesized that combination of neratinib with other HER2-targeted agents, such as trastuzumab and ado-trastuzumab emtansine (T-DM1), may provide better anti-tumor efficacy compared to neratinib alone. To address this, we have developed, under an IRB approved protocol, two NSCLC patient-derived xenograft (PDX) models bearing HER2 kinase domain mutations, DFCI-315 (ERBB2 P780_Y781insGSP) and DFCI-359 (ERBB2 755_757LREdelinsPR). These PDX lines were characterized by different tumor kinetics and doubling times of approximately 12 and 52 days, respectively. IHC analysis confirmed the expression, but not overexpression, of HER2 with H-scores of 70.0 and 54.2, respectively. Treatment with single agent neratinib (P.O. QD x 28 days, 40 mg/kg) was effective in both HER2 mutant lung models, leading to a significant tumor growth inhibition (TGI) with tumor stasis in DFCI-315 (p<0.0001) and tumor regression (TR) of >20% in DFCI-359 (p<0.05). DFCI-315 was insensitive to monotherapy with trastuzumab (I.P. 2x/week x 4 weeks, 20 mg/kg) but responded to monotherapy with T-DM1 (I.V. QWK x 4 weeks, 3 mg/kg) resulting in about 50% TGI (p<0.005). Both agents in combination with neratinib showed additional anti-tumor activity leading to TR in the DFCI-315 model (p<0.0001 vs vehicle), though difference from neratinib alone was not significant. In contrast, DFCI-359 model responded dramatically to neratinib plus trastuzumab combination starting from Day 5 of dosing, reaching TR of 62% by 3 weeks, compared to 25-30% TR by either of single agents (p<0.0001 vs vehicle, p<0.05 vs neratinib or trastuzumab). Tumor growth stasis following the combination treatment was durable for at least 10 days after the drug withdrawal, while the tumors regrew immediately after the drug withdrawal following neratinib treatment alone. Surprisingly, T-DM1 had no anti-tumor activity in this model and its combination with neratinib was indifferent from neratinib alone. Taken together, these data demonstrate neratinib has efficacy as single agent in preclinical models of HER2 mutant NSCLC and its anti-tumor activity was enhanced when combined with trastuzumab. Neratinib plus trastuzumab is a promising combination treatment for HER2 mutant lung cancer and is currently under clinical investigation (ClinicalTrials.gov NCT01953926).
Citation Format: Mari Kuraguchi, Luke J. Taus, Shan Zhou, Francesca Avogadri-Connors, Richard E. Cutler, Alshad S. Lalani, Paul Kirschmeier, Pasi A. Jänne. Exploring optimal targeted combination therapies with neratinib for HER2 mutated NSCLC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4806.
Abstract
As immune checkpoint blocking antibodies increasing become foundational therapies for the treatment of cancer, there is a pressing need to identify compounds that synergize with checkpoint ...blockade as the basis of combinatorial treatment regimens. We have developed a screening assay in which a luciferized tumor cell line expressing a model antigen is co-cultured with a transgenic CD8+ T cell specifically recognizing the model antigen in a H-2b-restricted manner. The target tumor cell/T cell assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells in an antigen-dependent manner. The EGFR inhibitor Erlotinib was the top hit that enhanced T cell killing of tumor cells. Subsequent experiments with Erlotinib and additional EGFR inhibitors validated the screen result. EGFR inhibitors increase both basal and IFN-γ-induced antigen processing and presentation of MHC class-I, which enhanced recognition and lysis by CD8+ cytotoxic T lymphocytes. The tumor cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen utilizing the same target tumor cell/T cell assay identified sgRNAs targeting EGFR as sensitizing tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in the MC38 syngeneic colon cancer model that was superior to PD-1 blockade or EGFR inhibition alone, further validating EGFR inhibitors as immunomodulatory agents that enhance PD-1 checkpoint blockade. This novel target tumor cell/T cell assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds AND target genes, respectively, that enhance or inhibit T cell recognition and killing of tumor cells.
Citation Format: Patrick H. Lizotte, Troy Luster, Megan E. Cavanaugh, Luke J. Taus, Abha Dhaneshwar, Naomi Mayman, Aaron Yang, Mark Bittinger, Paul Kirschmeier, Nathanael S. Gray, David A. Barbie, Pasi A. Janne. High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4935.
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