Small Cell Lung Cancer Patient-derived Xenograft Research Articles

Molecular phenotyping of small cell lung cancer using targeted cfDNA profiling of transcriptional regulatory regions.

We report an approach for cancer phenotyping based on targeted sequencing of cell-free DNA (cfDNA) for small cell lung cancer (SCLC). In SCLC, differential activation of transcription factors (TFs), such as ASCL1, NEUROD1, POU2F3, and REST defines molecular subtypes. We designed a targeted capture panel that identifies chromatin organization signatures at 1535 TF binding sites and 13,240 gene transcription start sites and detects exonic mutations in 842 genes. Sequencing of cfDNA from SCLC patient-derived xenograft models captured TF activity and gene expression and revealed individual highly informative loci. Prediction models of ASCL1 and NEUROD1 activity using informative loci achieved areas under the receiver operating characteristic curve (AUCs) from 0.84 to 0.88 in patients with SCLC. As non-SCLC (NSCLC) often transforms to SCLC following targeted therapy, we applied our framework to distinguish NSCLC from SCLC and achieved an AUC of 0.99. Our approach shows promising utility for SCLC subtyping and transformation monitoring, with potential applicability to diverse tumor types.

Abstract 606: The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with rapid disease progression and poor patient survival. Therapeutic options for SCLC remain a challenge due to limited understanding of both the biology of the disease and the major determinants of treatment response. Key transcription factors associated with unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Previously, we demonstrated that onvansertib, a highly specific inhibitor of the PLK1 Ser/Thr kinase, was effective in SCLC preclinical models, leading to its ongoing clinical development for relapsed/refractory SCLC patients (NCT05450965). Here we further explored potential strategies to enhance onvansertib activity by assessing its in vitro and in vivo activity in combination treatments. Methods: Human SCLC cell lines were treated for 72 hr with onvansertib in combination with paclitaxel, lurbinectedin or the ATR inhibitors BAY1895344 and AZD6738. Effect on cell proliferation was analyzed by WST1 and CellTiterGlo assays. In vivo activity of onvansertib in combination with paclitaxel was tested in 3 SCLC patient-derived xenograft (PDX) models either cisplatin-sensitive (TKO5- ASCL1 subtype) or cisplatin-resistant (TKO2-ASCL1 subtype, TKO8-NEUROD1 subtype). Tumor-bearing nude mice were treated with vehicle, onvansertib (50 mg/kg, oral, 4 times/week), paclitaxel (15 mg/kg, IP, once a week) or the combination for 5 weeks. Mice were followed for tumor growth and survival (defined as time to reach tumor volume greater than 2000 mm3). Results: The combination of onvansertib and paclitaxel inhibited cell proliferation synergistically in cell lines from 3 SCLC subtypes (ASCL1, NEUROD1, and YAP1), while the combinations with lurbinectedin or ATR inhibitors were not synergistic. We further examined the combination of onvansertib and paclitaxel in SCLC PDXs. The combination exhibited potent anti-tumor activity, resulting in significantly greater tumor growth inhibition and increase in survival compared to the monotherapies in all 3 PDXs. The combination was well tolerated with no observed body weight loss. While onvansertib and paclitaxel had negligible single agent activity in cisplatin-resistant TK02 PDX and TKO8 PDX models, the combination induced durable complete responses in 25% and 100% of treated mice respectively. Conclusion: The PLK1 inhibitor onvansertib in combination with paclitaxel showed synergy in multiple SCLC cell lines in vitro and potent anti-tumor activity in cisplatin-sensitive and -resistant SCLC PDX models in vivo. Tumor tissue analysis is underway to detail the mechanisms of this interaction. Citation Format: John C. Schmitz, Guojing Zhang, Abbe Pannucci, Tod Smeal, Chu-Chiao Wu, Maya Ridinger, Taofeek K. Owonikoko. The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 606.

Abstract LB_A22: Advancing personalized therapies for small cell lung cancer: Exploring HER2-targeting antibody-drug conjugates (ADCs) and SLFN11 biomarker insights

Abstract Background: Small-cell lung cancer (SCLC) is a highly aggressive malignancy constituting 15% of all lung cancers, with a 5-year survival rate of less than 7%. Our group has established four transcriptionally defined subtypes of SCLC, each with distinct cell surface targets (Gay et al, Cancer Cell 2021). Further, we identified SLFN11 (a putative DNA/RNA helicase) as a candidate biomarker of sensitivity to DNA-damaging agents, including topoisomerase I inhibitors. Despite these advances, SCLC is still treated clinically with a “one-size-fits-all” approach. Human epidermal growth factor receptor 2 (HER2/ERBB2) has been previously described in a subset of SCLCs and has been linked to poor prognosis. Notably, HER2-targeting antibody-drug conjugates (ADCs) like Trastuzumab Deruxtecan (T-DXd) (where the payload is a topoisomerase I inhibitor) have received FDA approval for various solid tumors, highlighting their therapeutic potential. Methods: We analyzed ERBB2 mRNA expression in publicly available patient datasets and profiled HER2 protein expression in cell lines and patient-derived xenograft models by flow cytometry and IHC. We then assessed the cytotoxicity of Trastuzumab Deruxtecan in a panel of cell lines representative of the heterogeneity of SCLC to test whether levels of HER2 and/or SLFN11 predicted response. Results: Analyses of HER2/ERBB2 in SCLC cell lines and patient-derived xenograft (PDX) models revealed an enrichment in the SCLC-POU2F3 (SCLC-P) subtype. HER2 expression was validated by IHC and flow cytometry in a subset of SCLC cell lines and PDX models. ERBB2 mRNA expression data was analyzed using publicly available SCLC patient datasets, where we uncovered high expression of ERBB2 in a subset of patient tumors. Consistent with this, single-cell RNA-Seq data revealed ERBB2 expression in POU2F3+ cells in tumors from patients with relapsed SCLC. As predicted, we found a strong correlation between the in vitro cytotoxicity of Trastuzumab Deruxtecan and both the level of surface HER2 expression and SLFN11 level. Importantly, the SCLC cell lines with the highest HER2 surface expression exhibited the lowest IC50 of Trastuzumab Deruxtecan, and the response was predicted by the presence of SLFN11. Conclusion: We demonstrate HER2/ERBB2 mRNA and protein expression in a subset of SCLC, with enrichment in the SCLC-P subtype. Further, the FDA-approved HER2-targeting ADC Trastuzumab Deruxtecan had significant cytotoxicity, correlating with expression of the surface target (HER2) as well as with SLFN11 (a candidate predictive biomarker for the topoisomerase inhibitor payload). These findings suggest the potential of tailoring HER2-targeting strategies based on HER2 and SLFN11 expression in SCLC as an effective and personalized approach for tackling SCLC. Citation Format: Ali H. Ibrahim, Bingnan Zhang, C. Allison Stewart, Alejandra Serrano, Luisa M. Solis, Wei-Lien Wang, Kavya Ramkumar, Robert J. Cardnell, Runsheng Wang, Alberto Duarte Jr, Lixia Diao, Yuanxin Xi, Jing Wang, Lauren A. Byers, Carl M. Gay. Advancing personalized therapies for small cell lung cancer: Exploring HER2-targeting antibody-drug conjugates (ADCs) and SLFN11 biomarker insights [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A22.

De Novo and Histologically Transformed Small-Cell Lung Cancer Is Sensitive to Lurbinectedin Treatment Through the Modulation of EMT and NOTCH Signaling Pathways.

Small-cell lung cancer (SCLC) is a high-grade neuroendocrine tumor with dismal prognosis and limited treatment options. Lurbinectedin, conditionally approved as a second-line treatment for metastatic SCLC, drives clinical responses in about 35% of patients, and the overall survival (OS) of those who benefit from it remains very low (∼9.3 months). This finding highlights the need to develop improved mechanistic insight and predictive biomarkers of response. We used human and patient-derived xenograft (PDX)-derived SCLC cell lines to evaluate the effect of lurbinectedin in vitro. We also demonstrate the antitumor effect of lurbinectedin in multiple de novo and transformed SCLC PDX models. Changes in gene and protein expression pre- and post-lurbinectedin treatment was assessed by RNA sequencing and Western blot analysis. Lurbinectedin markedly reduced cell viability in the majority of SCLC models with the best response on POU2F3-driven SCLC cells. We further demonstrate that lurbinectedin, either as a single agent or in combination with osimertinib, causes an appreciable antitumor response in multiple models of EGFR-mutant lung adenocarcinoma with histologic transformation to SCLC. Transcriptomic analysis identified induction of apoptosis, repression of epithelial-mesenchymal transition, modulation of PI3K/AKT, NOTCH signaling associated with lurbinectedin response in de novo, and transformed SCLC models. Our study provides a mechanistic insight into lurbinectedin response in SCLC and the first demonstration that lurbinectedin is a potential therapeutic target after SCLC transformation.

Targeting BCL2 Overcomes Resistance and Augments Response to Aurora Kinase B Inhibition by AZD2811 in Small Cell Lung Cancer.

Therapeutic resistance to frontline therapy develops rapidly in small cell lung cancer (SCLC). Treatment options are also limited by the lack of targetable driver mutations. Therefore, there is an unmet need for developing better therapeutic strategies and biomarkers of response. Aurora kinase B (AURKB) inhibition exploits an inherent genomic vulnerability in SCLC and is a promising therapeutic approach. Here, we identify biomarkers of response and develop rational combinations with AURKB inhibition to improve treatment efficacy. Selective AURKB inhibitor AZD2811 was profiled in a large panel of SCLC cell lines (n = 57) and patient-derived xenograft (PDX) models. Proteomic and transcriptomic profiles were analyzed to identify candidate biomarkers of response and resistance. Effects on polyploidy, DNA damage, and apoptosis were measured by flow cytometry and Western blotting. Rational drug combinations were validated in SCLC cell lines and PDX models. AZD2811 showed potent growth inhibitory activity in a subset of SCLC, often characterized by, but not limited to, high cMYC expression. Importantly, high BCL2 expression predicted resistance to AURKB inhibitor response in SCLC, independent of cMYC status. AZD2811-induced DNA damage and apoptosis were suppressed by high BCL2 levels, while combining AZD2811 with a BCL2 inhibitor significantly sensitized resistant models. In vivo, sustained tumor growth reduction and regression was achieved even with intermittent dosing of AZD2811 and venetoclax, an FDA-approved BCL2 inhibitor. BCL2 inhibition overcomes intrinsic resistance and enhances sensitivity to AURKB inhibition in SCLC preclinical models.

Abstract ND10: Discovery of MRT-2359, an orally bioavailable GSPT1 molecular glue degrader, for MYC-driven cancers

Abstract MYC transcription factors have been demonstrated to be drivers of many cancers, yet have remained recalcitrant to new drug development. We hypothesized that MYC-driven cancer cell lines are addicted to protein translation, and therefore are vulnerable to the loss of the translation termination factor, GSPT1. We have discovered MRT-2359, a selective GSPT1 molecular glue degrader, that demonstrates potent and preferential antiproliferative activity in MYC-driven cell lines, such as high N- and L-MYC mRNA expressing non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) lines. MRT-2359 has been optimized for differential toxicity between MYC- and non-MYC-driven cancer cell lines, kinetics and depth of degradation, oral bioavailability, and in vivo efficacy. The anti-tumor activity of MRT-2359 was assessed in more than 80 lung patient-derived xenografts (PDXs) that confirmed the preferential anti-tumor activity in N- and L-MYC high NSCLC and SCLC PDXs when dosed orally daily or intermittently. Similar levels of activity were also observed in neuroendocrine lung cancer PDXs and lymphoma models. Oral MRT-2359 is currently in a Phase 1/2 clinical trial in selected cancer patients with MYC-driven NSCLC, SCLC, high grade neuroendocrine cancers and diffuse large B-cell lymphoma (NCT05546268). Citation Format: Owen B. Wallace, Gerald Gavory, Mahmoud Ghandi, Anne-Cecile d’Alessandro, Debora Bonenfant, Maciej Cabanski, Lisa Cantagallo, Agustin Chicas, Qian Chen, Anna Diesslin, Christopher King, Vittoria Massafra, Rajiv Narayan, Arnaud Osmont, Dave Peck, Carolina Perdomo Ortiz, Martin Schillo, Ambika Singh, Ralph Tiedt, Simone Tortoioli, Silvia Buonamici, Markus Warmuth, Filip Janku, Bernhard Fasching. Discovery of MRT-2359, an orally bioavailable GSPT1 molecular glue degrader, for MYC-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND10.

Exportin 1 as a therapeutic target against chemoresistance in small cell lung cancer.

e20613 Background: Small cell lung cancer (SCLC) is an extremely aggressive disease comprising around 13% of lung cancer cases and responsible for approximately 250,000 deaths globally per year. A major cause of SCLC poor prognosis is the sparse treatment options available, typically resulting in only transient responses. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy to first-line chemotherapy showing only limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. Methods: We performed an in vitro CRISPR screen in SCLC cell lines to identify potential therapeutic targets to sensitize to chemotherapy, including short-term cultured cell lines derived from patient-derived xenografts (PDXs). Candidate hits were validated genetically and pharmacologically with in vitro and in vivo. Signaling pathways involved in phenotypes observed were studied by RNA sequencing and western blot, and toxicity studies were performed in vivo to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs). Results: Our CRISPR screen revealed the nuclear exporter XPO1 (Exportin 1) as a promising target sensitizing to chemotherapy, independently of the SCLC subtype. Importantly, exportin 1 is a therapeutic target in hematological malignancies, counting with a potent and selective inhibitor, selinexor, approved for clinical use. Combination of selinexor with cisplatin or irinotecan demonstrated high synergy in vitro and exquisite efficacy i n vivo in an array of chemonäive and chemoresistant SCLC PDXs, respectively, including all major SCLC subtypes. These chemotherapy-sensitizing effects were associated with the ability of Exportin 1 to impair chemotherapy-induced AKT overactivation, potentially occurring as a cytoprotective/anti-apoptotic mechanism in response to chemotherapy. The combinations were well tolerated in mice. We found that XPO1 mRNA expression was higher in SCLC than in any other solid tumor type or hematological malignancies, which we were able to confirm at the protein level in clinical TMAs. Conclusions: Exportin 1 inhibition strongly enhances sensitivity of SCLC tumors to cisplatin and irinotecan, used in first line and second line treatment of SCLC tumors, respectively. These results provide preclinical rationale for the combination of selinexor with first line and second line chemotherapy in SCLC. The clinical availability of selinexor will allow immediate clinical translation of these results in a disease setting with extremely limited therapeutic options.

CD133 as a Biomarker for an Autoantibody-to-ImmunoPET Paradigm for the Early Detection of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a deadly neuroendocrine tumor for which there are no screening methods sensitive enough to facilitate early, effective intervention. We propose targeting the neuroendocrine tumor neoantigen CD133 via antibody-based early detection and PET (immunoPET) to facilitate earlier and more accurate detection of SCLC. Methods: RNA sequencing datasets, immunohistochemistry, flow cytometry, and Western blots were used to quantify CD133 expression in healthy and SCLC patients. CD133 was imaged invivo using near-infrared fluorescence (NIRF) immunoimaging, and 89Zr immunoPET. Anti(α)-CD133 autoantibody levels were measured in SCLC patient plasma using antibody microarrays. Results: Across 6 publicly available datasets, CD133 messenger RNA was found to be higher in SCLC tumors than in other tissues, including healthy or normal adjacent lung and non-SCLC samples. Critically, the upregulation of CD133 messenger RNA in SCLC was associated with a significant increase (hazard ratio, 2.62) in death. CD133 protein was expressed in primary human SCLC, in SCLC patient-derived xenografts, and in both SCLC cell lines tested (H82 and H69). Using an H82 xenograft mouse model, we first imaged CD133 expression with NIRF. Both invivo and ex vivo NIRF clearly showed that a fluorophore-tagged αCD133 homed to lung tumors. Next, we validated the noninvasive visualization of subcutaneous and orthotopic H82 xenografts via immunoPET. An αCD133 antibody labeled with the positron-emitting radiometal 89Zr demonstrated significant accumulation in tumor tissue while producing minimal uptake in healthy organs. Finally, plasma αCD133 autoantibodies were found in subjects from cohort studies up to 1 year before SCLC diagnosis. Conclusion: In light of these findings, we conclude that the presence of αCD133 autoantibodies in a blood sample followed by CD133-targeted 89Zr-immunoPET could be an effective early detection screening strategy for SCLC.

Targeting the Ubiquitin-Proteasome System Using the UBA1 Inhibitor TAK-243 is a Potential Therapeutic Strategy for Small-Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive disease with an overall 5-year survival rate of less than 10%. Treatment for SCLC with cisplatin/etoposide chemotherapy (C/E) ± radiotherapy has changed modestly over several decades. The ubiquitin-proteasome system is an underexplored therapeutic target for SCLC. We preclinically evaluated TAK-243, a first-in-class small molecule E1 inhibitor against UBA1. We assessed TAK-243 in 26 SCLC cell-lines as monotherapy and combined with C/E, the PARP-inhibitor, olaparib, and with radiation using cell viability assays. We interrogated TAK-243 response with gene expression to identify candidate biomarkers. We evaluated TAK-243 alone and in combination with olaparib or radiotherapy with SCLC patient-derived xenografts (PDX). Most SCLC cell lines were sensitive to TAK-243 monotherapy (EC50 median 15.8 nmol/L; range 10.2 nmol/L-367.3 nmol/L). TAK-243 sensitivity was associated with gene-sets involving the cell cycle, DNA and chromatin organization, and DNA damage repair, while resistance associated with cellular respiration, translation, and neurodevelopment. These associations were also observed in SCLC PDXs. TAK-243 synergized with C/E and olaparib in vitro across sensitive and resistant SCLC cell lines. Considerable TAK-243-olaparib synergy was observed in an SCLC PDX resistant to both drugs individually. TAK-243 radiosensitization was also observed in an SCLC PDX. TAK-243 displays efficacy in SCLC preclinical models. Enrichment of gene sets is associated with TAK-243 sensitivity and resistance. TAK-243 exhibits synergy when combined with genotoxic therapies in cell lines and PDXs. TAK-243 is a potential therapeutic strategy to improve SCLC patient outcomes, both as a single agent and in combination with existing therapies.

Abstract P079: The impact of BCL2 expression on sensitivity to the novel Aurora kinase B inhibitor AZD2811 in small cell lung cancer

Abstract Purpose: Small cell lung cancer (SCLC) is a highly lethal malignancy, with rapidly-acquired therapeutic resistance. In contrast to non-SCLC, treatment strategies based on molecular subtypes have not been well established. Aurora kinase family proteins (AURKA and AURKB) are essential for cell division, regulating chromosomal segregation during mitosis, and are upregulated in cancer. Our group has demonstrated that cMYC-driven SCLC tumors were susceptible to an AURKA inhibitor, alisertib, making AURK proteins an attractive targeted therapeutic approach. A novel AURKB inhibitor, AZD2811NP (nanoparticle), is now being investigated in relapsed SCLC patients (NCT02579226), but molecular mechanisms of resistance have not yet been identified. Here, we hypothesize that targeting predictive markers related to the effect of AZD2811 may reinforce susceptibility to AURKB inhibition. Experimental Design: We evaluated susceptibility to AZD2811 in 63 human-derived SCLC cell lines using 96-hour proliferation assays. To identify translatable biomarkers of response, we correlated AZD2811 IC50 values with genomic (whole exome sequencing, WES), transcriptomic (RNASeq), and proteomic profiling (Reverse Phase Protein Array, RPPA) data. We validated changes in apoptosis and DNA damage markers induced by AZD2811 in SCLC cells infected with the lentivirus vectors expressing BCL-2 and shRNA against BCL-2 using western blot. We used SCLC patient-derived xenograft (PDX) models with distinct BCL-2 profiles to evaluate in vivo antitumor effect. Results: In the SCLC cells treated with AZD2811, 15/63 (24%) and 10/63 (16%) cell lines showed high sensitivity (IC50<30 nM, Cmax) and intermediate sensitivity (IC50=30-100nM). Comparing protein expression, we found that cMYC (Fold change, FC:2.5; P = 0.015) were a positive biomarker of sensitivity, while high BCL-2 (FC:1.86; P = 0.032) associated with resistance. cMYC-high SCLC cell lines that were susceptible to AZD2811 became resistant when BCL-2 was overexpressed. Conversely, BCL-2 knock-down enhanced response to AZD2811, inducing apoptosis and DNA damage, in BCL-2-high cells that were originally resistant to single-agent AZD2811. Similar to Bcl-2 knock-down, treatment with venetoclax (a BCL-2 inhibitor routinely used in other cancers) enhanced apoptosis and DNA damage induction in combination with AZD2811 in cells overexpressing BCL-2. In PDX models with high BCL-2, combination of AZD2811 and venetoclax prominently induced tumor regression and apoptosis compared with each monotherapy, while there was no significant difference in PDX models with low BCL-2 between the combination and the monotherapy. Conclusions: Our preclinical results indicate that high BCL-2 expression reduced the efficacy of AZD2811 in SCLC models, but that Bcl2-driven resistance could be overcome with the combined use of BCL-2 and AURKB inhibitors. Findings support a promising rational combination therapy for BCL-2-high SCLC to enhance response to aurora kinase B inhibition. Citation Format: Azusa Tanimoto, Carminia M. Della Corte, Kavya Ramkumar, Robert J. Cardnell, Allison C. Stewart, Carl M. Gay, Lauren Averett Byers, Qi Wang, Li Shen, Jing Wang, Jon Travers. The impact of BCL2 expression on sensitivity to the novel Aurora kinase B inhibitor AZD2811 in small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P079.

The First-in-Class UBA1 Inhibitor, TAK-243, in Combination With Radiotherapy for YAP1 and BEND3 Biomarker-Defined Small Cell Lung Cancer

The First-in-Class UBA1 Inhibitor, TAK-243, in Combination With Radiotherapy for YAP1 and BEND3 Biomarker-Defined Small Cell Lung Cancer

Identification and Characterization of the Molecular Mechanisms of SCLC Chemo-Radiation Resistance

Small cell lung cancer (SCLC) is among the most aggressive form of lung malignancies and accounts for 15-20% of all lung cancers. It has the tendency to metastasize early, thus limited-stage SCLC patients still receive systemic treatment with chemo-radiotherapy (chemoXRT) for their localized disease. SCLC is exceptionally sensitive to chemoXRT and exhibits high response rates; however, the recurrence rate is > 80% and patients relapse with tumors that resist further treatments. Elucidating mechanisms of chemoXRT resistance in SCLC is needed to develop improved therapies and positively impact patient outcomes.To better interrogate mechanisms of chemoXRT resistance, we developed a SCLC patient-derived xenograft (PDX) in vivo system for the major molecular subtypes of SCLC (classic and variant). We also established a novel PDX organoid (PDO) model to study the molecular underpinnings of chemoXRT resistance in SCLC.PDX tumor bearing mice were treated with: 1) vehicle control; 2) cisplatin plus etoposide (EP); 3) radiotherapy (XRT); and 4) both EP/XRT. A major response was observed within the EP/XRT arm compared to vehicle or single therapy arms. Whole transcriptome profiling among all treatment arms revealed molecular pathways and biological processes associated with chemoXRT resistance. By comparing our data with two previous SCLC patient cohort studies, we identified gene candidates for functional validation of chemoXRT resistance (i.e., ST6GAL1, TNIK and SOHLH2). To enable real-time cellular and molecular analysis of PDX behavior ex vivo and to validate SCLC chemoXRT resistance candidate genes, classic and variant SCLC organoids will be used. PDOs can be cultured up to 30 days in medium and parental PDX molecular subtype phenotypes are maintained as shown by comparative genomic hybridization, RNA-seq and protein marker analyses. Additionally, PDOs are capable of forming tumors in vivo.Genetically manipulated SCLC PDOs will be utilized as a tool to identify and validate candidates for chemoXRT resistance to be used as biomarkers and targets to combat chemoXRT resistance in SCLC.

MA16.03 CRISPR Screen Reveals XPO1 as a Therapeutic Target Strongly Sensitizing to First and Second Line Therapy in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an exceptionally aggressive disease comprising 13% of all lung cancer cases. With limited treatment options that typically result in transient responses, SCLC is responsible for approximately 250,000 deaths globally per year. The recent addition of immunotherapy to first-line platinum-based doublet chemotherapy appears to benefit only a small subset of patients, resulting in a 2-month increase in median overall survival. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. We performed an in vitro CRISPR screen in seven SCLC cell lines, including commercially available and short term-cultured cell lines derived from patient-derived-xenografts (PDXs), representing all major SCLC subtypes. We used an in-house druggable genome library including targets for which active inhibitors, either FDA-approved or in clinical development, are available. The screen was performed with two conditions per cell line, untreated and cisplatin-treated, to identify targets whose disruption would specifically sensitize to cisplatin. Candidate targets of interest were validated genetically with independent sgRNAs in Cas9-expressing in vitro and in vivo models (pSpCTRE-PDXs) and pharmacologically, with in vitro synergy assays and PDX treatments. Signaling pathways were studied by western blot, and toxicity studies were performed in vivo, to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in SCLC tissue microarrays (TMAs). Our CRISPR screen approach revealed XPO1 (Exportin 1) as a candidate that may contribute to cisplatin resistance across all SCLC subtypes. Combination of Exportin 1 inhibitors with cisplatin demonstrated synergy in vitro, and use of cisplatin with the Exportin 1 inhibitor selinexor showed combinatorial efficacy in vivo in chemonäive SCLC PDXs representing all SCLC subtypes. This efficacy was associated with increased DNA damage and apoptosis. The combination was well tolerated in mice at the drug concentrations tested. Exportin 1 inhibition also exhibited combinatorial synergy with irinotecan, a standard agent use for recurrent SCLC, and combination of selinexor with irinotecan demonstrated substantial efficacy in SCLC PDXs derived from chemorelapsed tumors including models for all SCLC subtypes, again with manageable toxicity profiles. We found SCLC to have the highest XPO1 mRNA expression among a diverse array of tumor histologies. High XPO1 expression was confirmed at the protein level in a SCLC tissue microarray, independent of SCLC subtype. Exportin 1 is highly expressed in SCLC tumors, independent of their subtype, and its inhibition enhances sensitivity to the chemotherapeutic drugs used in first line and second line treatment of SCLC tumors. Our results provide preclinical rationale for the combination of selinexor with cisplatin or with irinotecan as first or second line treatment, respectively, of SCLC.

Evaluation of the Small-molecule BRD4 Degrader CFT-2718 in Small-cell Lung Cancer and Pancreatic Cancer Models.

Targeted, catalytic degradation of oncoproteins using heterobifunctional small molecules is an attractive modality, particularly for hematologic malignancies, which are often initiated by aberrant transcription factors and are challenging to drug with inhibitors. BRD4, a member of the bromodomain and extraterminal family, is a core transcriptional and epigenetic regulator that recruits the P-TEFb complex, which includes Cdk9 and cyclin T, to RNA polymerase II (pol II). Together, BRD4 and CDK9 phosphorylate serine 2 (pSer2) of heptad repeats in the C-terminal domain of RPB1, the large subunit of pol II, promote transcriptional elongation. Small-molecule degraders of BRD4 have shown encouraging efficacy in preclinical models for several tumor types but less efficacy in other cancers including small-cell lung cancer (SCLC) and pancreatic cancer. Here, we evaluated CFT-2718, a new BRD4-targeting degrader with enhanced catalytic activity and in vivo properties. In vivo, CFT-2718 has significantly greater efficacy than the CDK9 inhibitor dinaciclib in reducing growth of the LX-36 SCLC patient-derived xenograft (PDX) model and performed comparably to dinaciclib in limiting growth of the PNX-001 pancreatic PDX model. In vitro, CFT-2718 reduced cell viability in four SCLC and two pancreatic cancer models. In SCLC models, this activity significantly exceeded that of dinaciclib; furthermore, CFT-2718 selectively increased the expression of cleaved PARP, an indicator of apoptosis. CFT-2718 caused rapid BRD4 degradation and reduced levels of total and pSer2 RPB1 protein. These and other findings suggest that BRD-mediated transcriptional suppression merits further exploration in the setting of SCLC.

Nelfinavir Inhibits the Growth of Small-cell Lung Cancer Cells and Patient-derived Xenograft Tumors.

Small-cell lung cancer (SCLC) is aggressive and confers poor prognosis. Although SCLC shows more response to chemotherapy than other types of lung cancer, it is difficult to cure because of its frequent recurrence. New drugs and molecular targets need to be identified. We investigated the effect of nelfinavir, an HIV protease inhibitor, on SCLC cells and in preclinical treatment studies using SCLC patient-derived xenograft (PDX) mouse models. Nelfinavir inhibited SCLC cell proliferation and induced cell death in vitro, which was caused by induction of the unfolded protein response (UPR), inhibition of mammalian/mechanistic target of rapamycin (mTOR) activation, and reduction in the expression of SCLC-related molecules such as achaete-scute homolog 1 (ASCL1). In vivo, nelfinavir inhibited the growth of SCLC PDX tumors, which correlated with the induction of UPR and reduced expression of ASCL1. Nelfinavir is highly effective in SCLC in vitro and in vivo, suggesting possible incorporation of nelfinavir into clinical trials for patients with SCLC.

Abstract 2692: Novel chemotherapy stable subpopulations are conserved across multiple small cell lung carcinoma patient derived xenograft models

Abstract Introduction: Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma of the lung responsible for up to 25% of lung cancer deaths and the 6th leading cause of cancer death. SCLC initially responds well to chemotherapy, but inevitably recurs even after initial complete responses. The etiology of this relapse is likely secondary to tumor heterogeneity and/or chemotherapy resistance subpopulations reconstituting tumor. Characterization of these resistant subpopulations could yield novel therapeutic targets for SCLC treatment. Mass cytometry uses metal labeled antibodies to profile expression and phosphorylation of multiple proteins in a single cell and offers the opportunity to identify new subpopulations as targets for novel therapies in SCLC. Methods: Nude mice with SCLC patient derived xenografts (PDXs) were treated with a single cycle of carboplatin/etoposide or saline injection. PDX samples were stained with a 26-35 marker panel and an intercalator dye to identify nucleated cells. This panel measured phospho-signaling, neuroendocrine, immune, and mesenchymal cell markers, and functional markers including ki67 and cleaved caspase 3. Mouse cells, including leukocytes, were excluded using mouse MHC1 gating and Histone H3 was used to identify nucleated cells Single cell protein expression and phosphorylation was analyzed using viSNE, manual gating, as well as unsupervised clustering approaches with SPADE to identified multiple subpopulations with neuroendocrine and non-neuroendocrine features. Subpopulations were compared across multiple patient derived xenograft (PDX) models with and without chemotherapy treatment. Results: Patient derived Xenograft (PDX) tumors across 3 distinct models including with and without a single cycle of chemotherapy treatment released viable tumor and stromal cells suitable for cryopreservation and mass cytometry. Chemotherapy treated tumors had dramatic changes in subpopulation distribution compared to matched mock treated tumor. This included enrichment in EPCAM+, CD24+, CD44- progenitor like subpopulations. Similar patterns of population shift were observed in multiple models. Of note, chemotherapy stable subpopulations were conserved across 5 different PDX models including SOX2+ and Oct ¾+ tumor populations. These subpopulations sorted similarly in multidimensional space in multiple PDX models suggesting conserved origins. Conclusions: Mass cytometry was able to identify multiple NE and non-neuroendocrine cell populations from SCLC PDXs and characterize their signaling include rare subpopulations with stem like signaling factors of interest. Chemotherapy treated PDX had differential subpopulation distribution with enrichment of progenitor like cells with chemotherapy treatment similar to previous work in mouse genetic SCLC. However, rare conserved chemotherapy stable subpopulations enriched in stem-like signaling factors were identified across 5 PDX models including those with and without chemotherapy treatment. This work raises the possibility that chemotherapy stable subpopulations may contribute to progenitor populations which lead to relapse. Future work in this lab will focus on characterizing the stem like features of these subpopulations and their phenotypic dynamics and signaling. Citation Format: Jonathan M. Lehman, Jeremy Staub, Yong Zou, Jonathan Irish, Pierre Massion. Novel chemotherapy stable subpopulations are conserved across multiple small cell lung carcinoma patient derived xenograft models [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 2692.

Abstract C091: Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications

Abstract Introduction: CDK7 is a key regulator of transcription and cell cycle progression and has been implicated in multiple tumor types driven by aberrant transcriptional control (e.g. MYC-, ESR1-activation) and/or aberrant cell cycle control (e.g. RB1, CCNE1, CDKN2A alterations). SY-5609 is an oral, potent, and highly selective CDK7 inhibitor that is advancing through IND-enabling studies to support initiation of a planned Phase 1 oncology trial in early 2020. Here we report on the relationship between pharmacokinetics (PK), pharmacodynamics (PD), and tumor growth inhibition (TGI) in xenograft models of tumor types with transcriptional and/or cell cycle aberrations including high grade serous ovarian cancer (HGSOC), small cell lung cancer (SCLC), triple negative breast cancer (TNBC), and estrogen receptor positive breast cancer (ER+BC). Methods: The relationship between SY-5609 PK, PD, and TGI was evaluated in xenograft models of HGSOC (OVCAR3) and TNBC (HCC70) using once daily (QD) or twice daily (BID) dosing via oral gavage. SY-5609 TGI was evaluated as a single agent (SA) QD in patient-derived xenograft (PDX) models of HGSOC (n=3), SCLC (n=4), TNBC (n=4), and in combination with once weekly fulvestrant in ER+BC PDX models selected in vivo for resistance to the CDK4/6 inhibitor palbociclib (n=1) or resistance to both palbociclib and fulvestrant (n=1). Results: SY-5609 plasma exposure was dose proportional and did not accumulate after repeated therapeutic doses. Dose-dependent transcriptional responses in xenograft tissue were observed within 4 hours of SY-5609 dosing and were sustained for 24 hours. TGI was dose-dependent, with tumor regressions observed at doses significantly below the maximum tolerated dose (MTD). Similar TGI was seen when the same daily dose was administered either QD or BID suggesting that the effect was driven by overall exposure or minimum concentration. In HGSOC, SCLC, and TNBC PDX models, SA SY-5609 induced >50% TGI in all models tested (11/11), with 7/11 (64%) demonstrating robust anti-tumor activity (≥90% TGI or regression): 3/3 HGSOC, 2/4 SCLC, and 2/4 TNBC. In a palbociclib-resistant ER+BC PDX model, the combination of SY-5609 and fulvestrant induced significant TGI (89%), with no evident tumor regrowth up to 21 days after dosing cessation, distinguishing the observed effects from SY-5609 SA or fulvestrant SA. In a palbociclib and fulvestrant double-resistant ER+ BC PDX model, SY-5609 SA resulted in 33% TGI and fulvestrant SA had no activity. In contrast, the combination of SY-5609 and fulvestrant demonstrated significant TGI (68%; p<0.0001 vs fulvestrant SA), suggesting re-sensitization to fulvestrant. Conclusions: We have characterized PK, PD, and anti-tumor activity of the CDK7 inhibitor SY-5609 in a series of xenograft models. Exposure in plasma is dose-proportional and does not accumulate at therapeutic dose levels. SY-5609 induces dose-dependent transcriptional responses in tumor xenograft tissue and shows robust TGI, including regressions, in PDX models derived from multiple solid tumor indications. These results highlight the broad potential for SY-5609 across a variety of solid tumor types, including treatment resistant ER+BC, and support the development of SY-5609 in early phase clinical trials. Citation Format: Liv H Johannessen, Shanhu Hu, Nan Ke, Anthony D'Ippolito, Nisha Rajagopal, Jason Marineau, Anneli Savinainen, William Zamboni, Graeme Hodgson. Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C091. doi:10.1158/1535-7163.TARG-19-C091

P1.12-05 Microenvironment Characterization of Small Cell Lung Cancer Xenografts Implanted in Hematopoietic Humanized Mice

With the high mutational burden seen in small cell lung cancer (SCLC) there is the theoretical potential to improve SCLC outcome through immunotherapy. Recent immunotherapeutic clinical trials in SCLC have demonstrated promising results. However, to better understand the immune response and the potential role of immunotherapy in SCLC, immunocompetent models are needed. To this end, we have applied a hematopoietic humanized mouse model, Hu-CB-BRGS to investigate the mechanisms underlying SCLC immunotherapy and develop novel strategies to improve therapeutic efficacy. BALB/c-Rag2nulllll2rynullSIRPαNOD (BRGS) pups were humanized through transplantation of cord blood (CB)-derived CD34+ cells. SCLC flank tumors were initiated in BRGS mice using two characterized SCLC cell lines (CDX) and 1 patient derived xenograft (PDX). Upon verification of human T-cell chimerism in the Hu-CB-BRGS mice, SCLC tumors grown in BRGS mice were engrafted into the flanks of Hu-CB-BRGS mice by trocar transfer. Tumor growth was quantified by twice weekly measurement and harvested on reaching 1200 mm3. At harvest, tumor tissue as well as host immune organs (lymph node, spleen) were collected for immunological assessment. Humanized immune system and tumor were evaluated by flow cytometry and immunohistochemistry (IHC). Flank tumors from two CDX tumors (H82 and H187) and one PDX tumor (LX-95) were successfully developed in the Hu-CB-BRGS mice with take rates averaging 82%. SCLC tumor growth rate in Hu-CB-BRGS mice was comparable to that seen in BRGS mice. Although human T cells were well represented in the lymph nodes and spleens of the Hu-CB-BRGS mice, we detected very few tumor infiltrating immune cells in the engrafted SCLC tumors by IHC and flow cytometry as defined by CD45 and CD3, which is consistent with the observations in SCLC patient tumor tissue. Tumor cells, identified by EpCAM expression, expressed low levels of MHC class I, II and PD-L1. PD-1 was expressed by human T cells found in the lymph nodes and spleen of Hu-CB-BRGS mice, while the SCLC xenografts expressed varying levels. We demonstrate that both SCLC PDX and CDX tumors can be grown in the context of a humanized immune system within mouse recipients. SCLC Hu-CB-BRGS mice demonstrate persistence of human immune cells, including T cells and B cells in the immune organs. The xenograft tumor microenvironment included variable human immune infiltrates. The SCLC Hu-CB-BRGS mouse model may be a valuable preclinical platform for testing human specific immune-oncology therapeutics for SCLC patients.

Abstract CT198: Expansion of Phase I/II study of olaparib and temozolomide in patients with relapsed small cell lung cancer (SCLC) and co-clinical trial in patient-derived xenografts (PDXs) for biomarker discovery

Abstract Background: SCLC is an aggressive high-grade neuroendocrine malignancy. While SCLC is often highly sensitive to first line etoposide/platinum (EP), response rates to second line cytotoxic chemotherapy range ~10-30%. Inhibition of poly(ADP-ribose) polymerase (PARP) by olaparib (O) has shown activity in SCLC in preclinical studies, and may synergize with the alkylating agent temozolomide (T). We previously reported a confirmed objective response rate (ORR) of 34.5% among an initial 30-patient SCLC cohort treated with combination OT in a Phase I/II trial (Farago et al., ASCO 2018). Here we provide analysis after further expansion to a total of 50 patients (pts). Methods: Eligibility criteria included histologically/cytologically confirmed incurable SCLC which had progressed following ≥ 1 platinum-based chemotherapy. O (tablet formulation) and T were administered orally on days 1-7 of 21-day cycles, with a recommended phase 2 dose (RP2D) of O 200 mg BID and T 75 mg/m2 QD. Response assessments were performed every 6 weeks. The primary endpoint was objective response rate (ORR). PDXs were generated from a subset of patients prior to OT and at progression, and OT and EP activity were assessed in vivo in a panel of 32 PDXs in a co-clinical trial. Whole transcriptome expression analysis using RNA sequencing was performed in 31 models to identify candidate biomarkers of sensitivity and resistance, and lead candidates were further interrogated using immunoblotting and immunohistochemistry. Results: Among 50 enrolled pts, the median (m) age was 63 (range 39-85), m number of prior therapies was 2 (range 1-7), 40% were male, and 72% were platinum sensitive. The confirmed ORR was 41.7%. After a m follow up of 7.1 months (mo) among 22 surviving patients, the m progression-free survival (PFS) was 4.2 mo, m overall survival (OS) was 8.5 mo, and m duration of response (DoR) was 4.3 mo. The ORR among platinum-sensitive and platinum-resistant pts was 47.1% and 28.6%, respectively, with no significant differences in mPFS, mOS or mDOR. The most common grade 3/4 treatment related adverse events were neutropenia (38%), anemia (28%) and thrombocytopenia (26%). Among 41 pts treated at the RP2D, dose reductions occurred in 44% overall and 64% of those who received at least 3 cycles. OT activity was further assessed in SCLC PDXs. The efficacy of OT in PDXs derived from 4 patients on the trial mirrored that seen in the patients. Across a panel of 32 PDXs, sensitivity to OT only modestly correlated with sensitivity to EP (Pearson r=0.50 for best response and r=0.48 for time to progression). Candidate biomarkers for sensitivity and resistance to OT were identified in PDX models and validated in patient samples, and will be presented. Conclusions: OT is shows promising clinical activity in relapsed SCLC. A co-clinical trial in PDXs provides a powerful platform for assessment of cross-resistance to other therapies and identification of clinically relevant biomarkers. Clinical trials identifier NCT02446704. Citation Format: Anna F. Farago, Beow Yeap, Yin P. Hung, Marcello Stanzione, Jun Zhong, Sarah Phat, David T. Myers, Robert Morris, Marina Kem, Deepa Rangachari, David Barbie, Subba R. Digumarthy, Lecia V. Sequist, Aaron N. Hata, Mari Mino-Kenudson, Alice T. Shaw, Nicholas Dyson, Benjamin J. Drapkin. Expansion of Phase I/II study of olaparib and temozolomide in patients with relapsed small cell lung cancer (SCLC) and co-clinical trial in patient-derived xenografts (PDXs) for biomarker discovery [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 CT198.

Abstract 381: Combined inhibition of Bcl-2 and MCL-1 in small cell lung cancer (SCLC) is most effective in tumors with low Bcl-xL expression

Abstract Introduction: SCLC is an aggressive high-grade neuroendocrine malignancy in which targeting anti-apoptotic regulators such as Bcl-2 and Bcl-xL has shown efficacy in pre-clinical models but has not resulted in successful clinical trials (Rudin et al., Clin Cancer Res. 2012). Although SCLC cell-lines do not reflect the clinical impact of these inhibitors, patient-derived xenograft (PDX) models may more accurately recapitulate Bcl-2 family expression profiles and BH3 mimetic efficacy. One promising hypothesis is that the fellow anti-apoptotic protein MCL-1 rescues viability in the presence of Bcl-2/Bcl-xL antagonists. Here we evaluate the efficacy of the MCL-1 inhibitor S63845 in combination with a novel specific inhibitor of Bcl-2, BCL201/S55746, in SCLC patient-derived xenografts. Methods: BH3 mimetic compounds were tested for synergy in vitro in SCLC cell lines. A set of ten cell lines was chosen based on relative expression of BCL2, MCL1, and BCL2L1 (Bcl-xL) mRNA. Single agent and and pair-wise combinations of Bcl2 family inhibitors were compared in three-day growth inhibition assays. Loewe synergy scores were plotted versus Bcl2 family mRNA expression to identify the determinants of drug sensitivity. Based on the cell line synergy assays, a combination of BCL201/S55746 and S63845 was selected to test in PDX models of SCLC. Bcl-2 family expression was profiled across a panel of 37 SCLC PDX models generated at MGH by quantitative western blot, and standardized to the most sensitive SCLC cell line, NCI-H211. Ten models were selected based on absolute expression of Bcl-2, Bcl-xL and MCL-1. Mice were treated when subcutaneous tumors reached a volume of 400-800 cc, enabling precise measurement of tumor regression and time to tumor regrowth. Findings: Bcl-2 family dependency in SCLC cell lines was profiled with selective inhibitors as single agents or combinations. Maximum synergy was found between BCL201/S55746 and S63845 in cell lines with the highest Bcl-2:Bcl-xL expression ratio. Bcl-2 family expression was profiled across a panel of 37 PDX models of SCLC, and a representative set of 10 models was selected for in vivo testing. Consistent with cell line results, the two most sensitive models to BCL201/S55746+S63845 demonstrated the highest Bcl-2:Bcl-xL ratios, with moderate to high expression of MCL-1. In these models BCL201/S55746+S63845 resulted in a 44-70% tumor regression that was stable throughout 4 weeks of treatment. Efficacy was not dependent on MCL-1 expression, and was not strongly correlated with PDX sensitivity to platinum-etoposide. Conclusions: Combined inhibition of Bcl-2 with BCL201 and MCL-1 with S63845 is effective in SCLC tumors with relatively low Bcl-xL expression. This combination overcomes MCL-1 mediated resistance to Bcl-2 inhibitors, and represents a promising strategy to target anti-apoptotic dependency in SCLC. Citation Format: Benjamin J. Drapkin, Sneha Sanghavi, David T. Myers, Jun Zhong, Sarah Phat, Youzhen Wang, Ensar Halilovic, Javad Golji, Anna Farago, Erick Morris, Nicholas J. Dyson. Combined inhibition of Bcl-2 and MCL-1 in small cell lung cancer (SCLC) is most effective in tumors with low Bcl-xL expression [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 381.