Small Cell Lung Cancer Models Research Articles

CDK9 inhibition as an effective therapy for small cell lung cancer

Treatment-naïve small cell lung cancer (SCLC) is typically susceptible to standard-of-care chemotherapy consisting of cisplatin and etoposide recently combined with PD-L1 inhibitors. Yet, in most cases, SCLC patients develop resistance to first-line therapy and alternative therapies are urgently required to overcome this resistance. In this study, we tested the efficacy of dinaciclib, an FDA-orphan drug and inhibitor of the cyclin-dependent kinase (CDK) 9, among other CDKs, in SCLC. Furthermore, we report on a newly developed, highly specific CDK9 inhibitor, VC-1, with tumour-killing activity in SCLC. CDK9 inhibition displayed high killing potential in a panel of mouse and human SCLC cell lines. Mechanistically, CDK9 inhibition led to a reduction in MCL-1 and cFLIP anti-apoptotic proteins and killed cells, almost exclusively, by intrinsic apoptosis. While CDK9 inhibition did not synergise with chemotherapy, it displayed high efficacy in chemotherapy-resistant cells. In vivo, CDK9 inhibition effectively reduced tumour growth and improved survival in both autochthonous and syngeneic SCLC models. Together, this study shows that CDK9 inhibition is a promising therapeutic agent against SCLC and could be applied to chemo-refractory or resistant SCLC.

Distinct regulation of ASCL1 by the cell cycle and chemotherapy in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive and lethal malignancy. Achaete-scute homolog 1 (ASCL1) is essential for the initiation of SCLC in mice and the development of pulmonary neuroendocrine cells (PNECs) which are the major cells of origin for SCLC. However, the regulatory mechanism of ASCL1 in SCLC remains elusive. Here, we found that ASCL1 expression gradually increases as the tumors grow in a mouse SCLC model and is regulated by the cell cycle. Mechanistically CDK2-CyclinA2 complex phosphorylates ASCL1, which results in increased proteasome-mediated ASCL1 protein degradation by E3 ubiquitin ligase HUWE1 during mitosis. TCF3 promotes the multi-site phosphorylation of ASCL1 through the CDK2-CyclinA2 complex and the interaction between ASCL1 and TCF3 protects ASCL1 from degradation. The dissociation of TCF3 from ASCL1 during mitosis accelerates the degradation of ASCL1. In addition, chemotherapy drugs greatly reduce the transcription of ASCL1 in SCLC cells. Depletion of ASCL1 sensitizes SCLC cells to chemotherapy drugs. Together, our study demonstrates that ASCL1 is a cell-cycle regulated protein and provides a theoretical basis for applying cell-cycle related antitumor drugs in SCLC treatment. Implications: Our study revealed a novel regulatory mechanism of ASCL1 by cell cycle and chemotherapy drugs in SCLC. Treating SCLC patients with a combination of ASCL1-targeting therapy and chemotherapy drugs could potentially be beneficial.

Abstract NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer

Abstract NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer

Abstract 5795: Potent and selective degradation of KAT2A and KAT2B induces profound cell state changes and inhibits growth of AML, SCLC and NEPC model systems

Abstract Phenotypic plasticity, or the ability of cancer cells to switch between cell states, has recently been recognized as a key hallmark of cancer. Cancer cells can hijack normal developmental pathways, preventing terminal differentiation or causing de-differentiation or transdifferentiation to a more proliferative, plastic cell state similar to those found in early development. Using our proprietary AI/ML platform AURIGIN, we created a high-resolution atlas to define the drivers of normal cell states during human development. After mapping primary human tumors to the atlas, we identified the histone acetyltransferase KAT2A as a key driver of tumor cell plasticity in a subset of acute myeloid leukemias (AML), small cell lung cancer (SCLC) and neuroendocrine prostate cancer (NEPC). Targeting KAT2A was genetically validated in MOLM-13 (AML) cells, where knockdown resulted in significant cell growth inhibition and induction of cell state changes consistent with a more mature differentiation phenotype. Similarly, an early tool heterobifunctional degrader of KAT2A and KAT2B (AUR101) resulted in potent growth inhibition and granulocytic differentiation in MOLM-13 cells and in 5/12 primary AML patient samples grown ex vivo. Treatment of NCI-H1048 SCLC cells with AUR101 potently degraded KAT2A/B, induced a significant shift from a dedifferentiated cell state to a more differentiated epithelial cell state and potently inhibited cell growth both in vitro and in vivo. The strong biological validation of KAT2A/B in SCLC and AML tumors, led us to develop AUR1545, a novel sub-nanomolar degrader of KAT2A and KAT2B. AUR1545, with its improved drug like properties, was tested across representative models of AML, SCLC and NEPC. In vitro, treatment with AUR1545 inhibited proliferation and promoted profound cell state changes in MOLM-13 (AML) cells. In NCI-H1048 (SCLC), LASPC-01 (NEPC) cell lines as well as a primary NEPC organoid model, AUR1545 demonstrated potent antiproliferative effects. In vivo, AUR1545 significantly inhibited tumor growth in an NCI-H1048 xenograft model. Taken together, these data genetically and pharmacologically validate KAT2A and KAT2B as key drivers of cell state plasticity in SCLC, NEPC and AML tumors and point to their utility as novel targets in these aggressive, metastatic and drug resistant cancers. Citation Format: Kimberly S. Straley, James Neef, Ileana Antony-Debre, Maulasri Bhatta, Sambad Sharma, Sara Sinicropi-Yao, Joe DeBartolo, Andrew McRiner, Betty Chan, Henry Wilson, Christina S. Lee, Zied Boudhraa, Mohamed El Ezzy, Umar Sharif, Mark Bittinger, Laura Antipov, Thomas G. Graeber, Stephane de Botton, Katharine E. Yen, David S. Millan. Potent and selective degradation of KAT2A and KAT2B induces profound cell state changes and inhibits growth of AML, SCLC and NEPC model systems [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 5795.

Abstract 3142: NN3201, a novel c-Kit targeting ADC, exhibits robust preclinical anti-tumor efficacy in SCLC and GIST models

Abstract c-Kit (CD117) is a type III receptor tyrosine kinase (RTK), which is known to be activated upon binding to its cognate ligand stem cell factor (SCF) to induce critical signaling pathways involved in cell proliferation, survival, and differentiation. Indicative of poor prognosis, overexpression and activating mutations of c-Kit have been implicated in a variety of human cancers including small cell lung cancer (SCLC), gastrointestinal stromal tumors (GIST), and acute myelogenous leukemia (AML). While imatinib, a small molecule c-Kit inhibitor, has been a beneficial treatment for GIST, resistance eventually develops to the drug, moreover, shows limited efficacy in c-Kit overexpressing tumors such as SCLC. Herein, we developed NN3201, a novel c-Kit targeting antibody-drug conjugate (ADC), comprised of fully human 2G4 antibody with a picomolar binding affinity to c-Kit and cytotoxic monomethyl auristatin E (MMAE) as the payload. NN3201 displayed potent in vitro cytotoxicity in various wild-type and mutant c-Kit positive SCLC and GIST cell lines with IC50 in the nanomolar range. In addition, NN3201 was rapidly internalized and inhibited c-Kit-mediated signaling., NN3201 treatment resulted in the increased proportions of the sub G1 and G2/M in the c-Kit positive cancer cell lines, indicating that release of MMAE induced cell cycle arrest. Moreover, bystander effect was confirmed by co-culturing c-Kit high and negative cell lines. Most importantly, in mouse xenograft models, NN3201 showed a remarkable tumor growth inhibition of several wild type, mutant c-Kit positive, and imatinib resistant SCLC and GIST cell lines. Enhanced anti-tumor activity and delayed tumor growth were observed in an SOC-treated (etoposide and carboplatin) SCLC xenograft model, by NN3201 in comparison to second line SOC (topotecan or irinotecan). In the GLP study, repeated intravenous administration of NN3201 was well tolerated at all doses (0.5, 1, 2 mg/kg Q3Wx3) without unscheduled death. Dose-dependent, yet transient, cytotoxicity of NN3201 was observed in the bone marrow. We confirmed HNSTD (Highest Non-Severely Toxic Dose) of NN3201 to be 2 mg/kg through exploratory and GLP repeat dose toxicity studies as no serious irreversible toxicity was observed. Accumulating data suggest that NN3201 is a promising therapeutic alternative for the treatment of SCLC and GIST regardless of wild-type or activating mutations in c-Kit. Citation Format: Chansik Kim, Jin Gu Cho, TaeMin Wi, Jiwoo Moon, Han-Jik Ko, Jina Lee, Jin Woo Park, Yeonjy Lee, Sanghee Kim, Jin-Ock Kim, Jaeyoung Song, Sun-Hwa Lee, Sang Gyu Park. NN3201, a novel c-Kit targeting ADC, exhibits robust preclinical anti-tumor efficacy in SCLC and GIST models [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 3142.

Abstract 6554: Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with a very poor prognosis and limited therapeutic options. Repression of the MHC-I and reduced tumor infiltration of cytotoxic T-lymphocytes is often associated with immunotherapy resistance in SCLC. Lurbinectedin is FDA-approved as a second-line treatment for SCLC and we previously showed lurbinectedin as an effective therapeutic strategy in SCLC preclinical models. There are ongoing clinical trials combining immune checkpoint blockade with lurbinectedin in ES-SCLC. However, there is no insight into the effect of lurbinectedin on the immune microenvironment in SCLC. In this study, we evaluated the effect of lurbinectedin on the immune microenvironment and the anti-tumor effect with or without PD-L1 blockade. Results: To determine the effect of lurbinectedin in SCLC models, we treated immunocompetent flank RPP (Rb1, Trp53, and p130) and RPM (Rb1, Trp53, and MYCT58A) tumor-bearing mice with lurbinectedin (0.2 mg/kg, 1/7days) and/or anti-PD-L1 (300µg, 1/7days) for 3 weeks. Mice treated with anti-PD-L1 alone showed no anti-tumor response, and single-agent lurbinectedin caused a delay in tumor growth. However, 6 of the 10 mice treated with the combination of lurbinectedin and anti-PD-L1, had a complete response (100% reduction) and the other 4 had 90% tumor regression. Tumors were resected and analyzed by multicolor flow cytometry for changes in tumor-infiltrating lymphocytes (TILs). Furthermore, analysis of immune cells infiltrating into the tumors post- treatment showed significant induction of cytotoxic T-cells and a reduction of exhausted and regulatory T cells in the combination treatment arm. Similarly, pro-inflammatory M1 type macrophages and dendritic cells were increased, while immunosuppressive M2 type macrophages and MDSC cells were decreased. These effects are consistent with data showing that lurbinectedin treatment leads to an activation of the cGAS/STING pathway, type I/II interferons (IFNα/β), and pro-inflammatory chemokines (CCL5, CXCL10) in tumors. Interestingly, lurbinectedin treatment led to significant upregulation of mRNA and surface expression of MHC class-I genes (HLA-A/B/C) in vitro and in vivo. Finally, bulk RNA sequencing as well as RT-PCR of pre- and post-lurbinectedin treatment demonstrated an increase in DAMPs associated with immunogenic cell death. Conclusion: We provide the first mechanistic insight into the lurbinectedin-induced multimodal immune modulation in SCLC leading to a dramatic anti-tumor activity accompanied by the establishment of a strong anti-tumor immune microenvironment. Since lurbinectedin is already approved as a second-line agent in SCLC, our preclinical data provide a strong rationale for combining this regimen with inhibitors of the PD-L1 pathway and also highlight the immune subsets that the combination targets. Citation Format: Subhamoy Chakraborty, Yazhini Mahendravarman, Utsav Sen, Avisek Banerjee, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response 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 6554.

Abstract 6028: Preferential tumor-to-normal tissue biodistribution and single-dose efficacy with ABD147, a DLL3-targeted engineered antibody-based radiotherapeutic, in preclinical small cell lung cancer models

Abstract Targeted radiotherapies represent an emerging treatment modality for aggressive cancers with limited treatment options, such as small cell lung cancer (SCLC). ABD147 is a Delta Ligand 3 (DLL3)-targeted antibody conjugate designed to carry and deliver cytotoxic radioactive isotopes to DLL3-expressing tumor cells. DLL3 is commonly expressed on the cell surface of neuroendocrine cancer cells, including SCLC, but has limited and predominantly intracellular expression in non-malignant tissues. The ABD147 antibody specifically binds human DLL3 with high affinity and is internalized by DLL3-expressing cancer cells. To minimize radiation exposure to normal tissue, ABD147 has been engineered to clear quickly from the blood compartment. Using indium-111 to determine ABD147 pharmacokinetics and biodistribution in mice, we demonstrate rapid blood and normal tissue clearance of 111In-ABD147, as designed, following a single intravenous dose administration. However, 111In-ABD147 retains high tumor accumulation (activity concentration of up to 30% ID/g) in xenograft mouse models of SCLC despite low DLL3 surface antigen expression on tumor cells (≤ 3000 copies). 111In-ABD147 shows a favorable tumor-to-normal tissue distribution with predominant liver clearance. Following ABD147 therapeutic radioisotope delivery of actinium-225 or lutetium-177 (225Ac-ABD147, 177Lu-ABD147), we demonstrate single-dose tumor regression and dose-dependent sustained anti-tumor efficacy, corresponding to an extension of survival out to 84 days in multiple SCLC xenograft models. In summary, ABD147 can preferentially deliver radionuclides to DLL3-expressing tumor tissue while substantially reducing the systemic radioactive exposure typical of conventional IgG radioconjugates. Following promising results in preclinical models, including potent anti-tumor activity, 225Ac-ABD147 is now progressing into clinical development. Citation Format: Iva Kulic, Etienne S. Melese, Emma Cummins, Alex Mandel, Raja Viswas, Michael Abrams, Adam Judge. Preferential tumor-to-normal tissue biodistribution and single-dose efficacy with ABD147, a DLL3-targeted engineered antibody-based radiotherapeutic, in preclinical small cell lung cancer models [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 6028.

Abstract 6602: Targeting the SWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies

Abstract Background: Small cell lung cancer (SCLC) is a rapidly progressing subtype of lung cancer with a high growth rate and early metastasis propensity, often resulting in a more advanced disease stage at diagnosis. A recent transcriptome analysis of human SCLC tumors revealed that SCLC could be characterized by the expression pattern of certain transcription factors (TFs), including ASCL1 (achaete-scute family bHLH transcription factor 1) and POU2F3 (POU domain class 2 transcription factor 3), exemplifying SCLC as a TF-driven malignancy. ASCL1-driven SCLC (SCLC-A) manifests a neuroendocrine phenotype, while POU2F3-driven SCLC (SCLC-P) is characterized as a tuft-cell-like variant. Recent studies revealed that POU domain class 2 transcription factors uniquely rely on coactivators to achieve their lineage-defining functions in the tuft cell and B cell lineages. In tuft-cell-like SCLC cells, the coactivators of POU2F3 (POU2AF2 and POU2AF3) endow POU2F3 with a critical transactivation domain by forming a master regulator complex, which supports enhancer-mediated cancer-promoting gene activation in SCLC-P cells. This indicates a potential therapeutic vulnerability of tuft-cell-like SCLC whereby strategies aimed at blocking POU2F3-POU2AF2/3 function may lead to clinical benefit. Methods: We conducted a domain-targeted CRISPR screen to identify druggable targets for SCLC-P, followed by pharmacological validation in preclinical SCLC models. Multi-omics techniques, including ATAC-seq, ChIP-seq, RNA-seq, and RIME, were employed to elucidate the SWI/SNF complex's regulatory influence on the POU2F-POU2AF axis. Results: Here we identified that the POU2F3 molecular subtype of SCLC (SCLC-P) exhibits an exquisite dependence on the activity of the SWI/SNF epigenetic complex. SCLC-P cell lines were sensitive to low nanomolar levels of a SWI/SNF ATPase degrader when compared to other molecular subtypes of SCLC. Co-factors of POU2F were found to interact with components of the SWI/SNF complex. The POU2F3 transcription factor complex is evicted from chromatin upon SWI/SNF ATPase degradation. A novel, orally bioavailable SWI/SNF ATPase PROTAC degrader demonstrated preferential efficacy in SCLC-P relative to the SCLC-A subtype. The SWI/SNF ATPase PROTAC degrader did not alter normal tuft cell numbers in lung or colon, nor did it exhibit toxicity in mice. B cell malignancies which displayed a dependency on the POU2F co-factor, POU2AF1, were also remarkably sensitive to SWI/SNF ATPase degradation. In a POU2AF1-dependent, disseminated murine model of multiple myeloma, AU-24118 had an enhanced survival benefit as compared to pomalidomide, an approved treatment for multiple myeloma. Conclusions: Taken together, our studies suggest that POU2F-POU2AF driven malignancies have an intrinsic dependence on the SWI/SNF complex representing a striking therapeutic vulnerability. Citation Format: Lanbo Xiao, Tongchen He, Olaf Klingbeil, Young Eleanor, Xiaoli Wu, Yuanyuan Qiao, Abhijit Parolia, Sanjana Eyunni, Rahul Mannan, Somnath Mahapatra, NamHoon Kim, Heng Zheng, Fengyun Su, Xuhong Cao, Susanta Samajdar, Murali Ramachandra, Christopher R. Vakoc, Arul M. Chinnaiyan. Targeting the SWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies [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 6602.

Comprehensive analysis of transcription factor-based molecular subtypes and their correlation to clinical outcomes in small-cell lung cancer

Recent studies have reported the predictive and prognostic value of novel transcriptional factor-based molecular subtypes in small-cell lung cancer (SCLC). We conducted an in-depth analysis pairing multi-omics data with immunohistochemistry (IHC) to elucidate the underlying characteristics associated with differences in clinical outcomes between subtypes. IHC (n=252), target exome sequencing (n=422), and whole transcriptome sequencing (WTS, n=189) data generated from 427 patients (86.4% males, 13.6% females) with SCLC were comprehensively analysed. The differences in the mutation profile, gene expression profile, and inflammed signatures were analysed according to the IHC-based molecular subtype. IHC-based molecular subtyping, comprised of 90 limited-disease (35.7%) and 162 extensive-disease (64.3%), revealed a high incidence of ASCL1 subtype (IHC-A, 56.3%) followed by ASCL1/NEUROD1 co-expressed (IHC-AN, 17.9%), NEUROD1 (IHC-N, 12.3%), POU2F3 (IHC-P, 9.1%), triple-negative (IHC-TN, 4.4%) subtypes. IHC-based subtype showing high concordance with WTS-based subtyping and non-negative matrix factorization (NMF) clusterization method. IHC-AN subtype resembled IHC-A (rather than IHC-N) in terms of both gene expression profiles and clinical outcomes. Favourable median overall survival was observed in IHC-A (15.2 months) compared to IHC-N (8.0 months, adjusted HR 2.3, 95% CI 1.4-3.9, p=0.002) and IHC-P (8.3 months, adjusted HR 1.7, 95% CI 0.9-3.2, p=0.076). Inflamed tumours made up 25% of cases (including 53% of IHC-P, 26% of IHC-A, 17% of IHC-AN, but only 11% of IHC-N). Consistent with recent findings, inflamed tumours were more likely to benefit from first-line immunotherapy treatment than non-inflamed phenotype (p=0.002). This study provides fundamental data, including the incidence and basic demographics of molecular subtypes of SCLC using both IHC and WTS from a comparably large, real-world Asian/non-Western patient cohort, showing high concordance with the previous NMF-based SCLC model. In addition, we revealed underlying biological pathway activities, immunogenicity, and treatment outcomes based on molecular subtype, possibly related to the difference in clinical outcomes, including immunotherapy response. This work was supported by AstraZeneca, Future Medicine 2030 Project of the Samsung Medical Center [grant number SMX1240011], the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [grant number 2020R1C1C1010626] and the 7th AstraZeneca-KHIDI (Korea Health Industry Development Institute) oncology research program.

Evaluating CD133 as a Radiotheranostic Target in Small-Cell Lung Cancer.

Despite decades of work, small-cell lung cancer (SCLC) remains a frustratingly recalcitrant disease. Both diagnosis and treatment are challenges: low-dose computed tomography (the approved method used for lung cancer screening) is unable to reliably detect early SCLC, and the malignancy's 5 year survival rate stands at a paltry 7%. Clearly, the development of novel diagnostic and therapeutic tools for SCLC is an urgent, unmet need. CD133 is a transmembrane protein that is expressed at low levels in normal tissue but is overexpressed by a variety of tumors, including SCLC. We previously explored CD133 as a biomarker for a novel autoantibody-to-immunopositron emission tomography (PET) strategy for the diagnosis of SCLC, work that first suggested the promise of the antigen as a radiotheranostic target in the disease. Herein, we report the in vivo validation of a pair of CD133-targeted radioimmunoconjugates for the PET imaging and radioimmunotherapy of SCLC. To this end, [89Zr]Zr-DFO-αCD133 was first interrogated in a trio of advanced murine models of SCLC─i.e., orthotopic, metastatic, and patient-derived xenografts─with the PET probe consistently producing high activity concentrations (>%ID/g) in tumor lesions combined with low uptake in healthy tissues. Subsequently, a variant of αCD133 labeled with the β-emitting radiometal 177Lu─[177Lu]Lu-DTPA-A″-CHX-αCD133─was synthesized and evaluated in a longitudinal therapy study in a subcutaneous xenograft model of SCLC, ultimately revealing that treatment with a dose of 9.6 MBq of the radioimmunoconjugate produced a significant increase in median survival compared to a control cohort. Taken together, these data establish CD133 as a viable target for the nuclear imaging and radiopharmaceutical therapy of SCLC.

Abstract A019: ATR inhibitor efficacy depends on CD8+ T-cell recruitment and MHC class-I upregulation via intratumoral STING pathway activation in small cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Recent clinical trials of immune checkpoint blockade (ICB) combined with chemotherapy delivered only very modest benefits. Using genetic screens, we identified inhibition of G2/M cell cycle regulator, ataxia telangiectasia, and rad3 related (ATR), as a promising therapeutic target in small cell lung cancer (SCLC). In this study, we investigated the effect of targeting ataxia telangiectasia and rad3-related (ATR), the primary activator of the replication stress response, on the immune microenvironment in SCLC. We further confirm the efficacy of combining ATR inhibition with immune checkpoint blockade in SCLC. Methods: In this study, we performed genetic and pharmacological inhibition of ATR in a panel of human and murine SCLC cell lines. Furthermore, we investigated the effect of ATR inhibition either alone or in combination with PD-L1 blockade in multiple immunocompetent mouse models of SCLC. The downstream effects of ATR inhibition was assessed by bulk RNA sequencing, multicolor flow cytometry, western blot analysis and real-time qRT-PCR. The SCLC clinical samples from treatment naïve and patients treated with an ATR inhibitor we analysis by single cell and bulk RNA sequencing to ascertain the effect of ATR on immune subsets. Results: In multiple immunocompetent SCLC mouse models, ATR inhibition (ATRi) remarkably enhanced the anti-tumor effect of PD-L1 blockade. We next tested the ATR inhibition either alone or in combination with PD-L1 in the second-line regimen for SCLC. We observed that ATR inhibition in combination with PD-L1 blockade significantly reduced tumor volume and prolonged survival of aggressive mice models when compared to PD-L1 alone. Targeting ATR enhanced the expression of PD-L1, activated the cGAS/STING pathway, induced the expression of Type I and II interferon pathways, and caused significant infiltration of cytotoxic and memory/effector T-cells into tumors. Interestingly, ATRi also led to significant induction of MHC class I in SCLC in vitro and in vivo models. Single-cell analysis of patient samples confirms immunosuppressed phenotype in SCLC. Analysis of pre-and post-treatment clinical samples from a proof-of-concept study of a first-in-class ATR inhibitor, M6620 (VX970, berzosertib), and TOP1 inhibitor topotecan, in patients with relapsed SCLCs, validated the induction of MHC class I and interferon pathway genes, for the first time in this disease. Conclusion: Our findings highlight ATRi as a potentially transformative vulnerability of SCLC, paving the way for combination clinical trials with anti-PD-L1. We are the first to show that ATR inhibition can activate MHC class I in SCLC clinical samples. Given the increasing importance of immunotherapy for the management of SCLC and that ATR inhibitors are already in clinical trials, combining an ATR inhibitor with PD-L1 blockade may offer a particularly attractive strategy for the treatment of SCLC and contribute to the rapid translation of this combination into the clinic. Citation Format: Triparna Sen. ATR inhibitor efficacy depends on CD8+ T-cell recruitment and MHC class-I upregulation via intratumoral STING pathway activation in small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A019.

Genetically-engineered mouse models of small cell lung cancer: the next generation.

Small cell lung cancer (SCLC) remains the most fatal form of lung cancer, with patients in dire need of new and effective therapeutic approaches. Modeling SCLC in an immunocompetent host is essential for understanding SCLC pathogenesis and ultimately discovering and testing new experimental therapeutic strategies. Human SCLC is characterized by near universal genetic loss of the RB1 and TP53 tumor suppressor genes. Twenty years ago, the first genetically-engineered mouse model (GEMM) of SCLC was generated using conditional deletion of both Rb1 and Trp53 in the lungs of adult mice. Since then, several other GEMMs of SCLC have been developed coupling genomic alterations found in human SCLC with Rb1 and Trp53 deletion. Here we summarize how GEMMs of SCLC have contributed significantly to our understanding of the disease in the past two decades. We also review recent advances in modeling SCLC in mice that allow investigators to bypass limitations of the previous generation of GEMMs while studying new genes of interest in SCLC. In particular, CRISPR/Cas9-mediated somatic gene editing can accelerate how new genes of interest are functionally interrogated in SCLC tumorigenesis. Notably, the development of allograft models and precancerous precursor models from SCLC GEMMs provides complementary approaches to GEMMs to study tumor cell-immune microenvironment interactions and test new therapeutic strategies to enhance response to immunotherapy. Ultimately, the new generation of SCLC models can accelerate research and help develop new therapeutic strategies for SCLC.

Abstract B027: ATR inhibition upregulates PD-L1 and potentiates the antitumor immune response to chemoimmunotherapy in small-cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with a very poor prognosis and limited therapeutic options. Recent clinical trials of immune checkpoint blockade (ICB) combined with chemotherapy delivered only very modest benefit. Here, we identified that ataxia telangiectasia and rad3 related (ATR), the primary activator of the replication stress response, is highly enriched in SCLC and inhibition of ATR induced DNA damage and apoptosis in multiple SCLC models. Methods: In this study, we performed genetic and pharmacological inhibition of ATR in a panel of human and murine SCLC models. Furthermore, we investigated the effect of ATR inhibition either alone or in combination with PD-L1 blockade either as a first-line or a second-line treatment in multiple immunocompetent mouse models of SCLC. The downstream effects of ATR inhibition was assessed by single-cell RNA sequencing, multicolor flow cytometry, western blot analysis, IHC, and genomic analysis. Results: In multiple immunocompetent SCLC mouse models, ATR inhibition (ATRi) remarkably enhanced the anti-tumor effect of PD-L1 blockade. We next tested the ATR inhibition either alone or in combination with PD-L1 in the second-line regimen for SCLC. We observed that ATR inhibition in combination with PD-L1 blockade significantly reduced tumor volume and prolonged survival of aggressive mice models when compared to PD-L1 alone. Targeting ATR enhanced the expression of PD-L1, activated the cGAS/STING pathway, induced the expression of Type I and II interferon pathways, and caused significant infiltration of cytotoxic and memory/effector T-cells into tumors. Interestingly, ATRi also led to significant induction of MHC class I in SCLC in vitro and in vivo models. Analysis of pre- and post-treatment clinical samples from a proof-of-concept study of a first-in-class ATR inhibitor, M6620 (VX970, berzosertib), and TOP1 inhibitor topotecan, in patients with relapsed SCLCs validated the induction of MHC class I and interferon pathway genes, for the first time in this disease. Conclusion: Our findings highlight ATRi as a potentially transformative vulnerability of SCLC, paving the way for combination clinical trials with anti-PD-L1. Given the increasing importance of immunotherapy for the management of SCLC and that ATR inhibitors are already in clinical trials, combining an ATR inhibitor with PD-L1 blockade may offer a particularly attractive strategy for the treatment of SCLC and contribute to the rapid translation of this combination into the clinic. Citation Format: Triparna Sen. ATR inhibition upregulates PD-L1 and potentiates the antitumor immune response to chemoimmunotherapy in small-cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B027.

Abstract B028: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a poorly immunogenic, high-grade neuroendocrine carcinoma arising in the lung. Immune checkpoint blockade (ICB) added to chemotherapy is now the standard upfront therapy for SCLC but leads to a modest increase in overall survival and progression-free survival. These modest benefits underscore the critical need to identify pathways and targets that can durably enhance the antitumor responses of ICB in SCLC. SCLC is characterized by a ubiquitous loss of TP53 which disrupts the G1-S cell cycle checkpoint and as a result, most SCLC is dependent on G2-M cell cycle checkpoint regulators including WEE1. We and others have shown that DNA damage response (DDR) components are overexpressed in SCLC and targeting DDR could be an effective therapeutic strategy in SCLC. Methods: In this study, we have performed WEE1 inhibition either alone or in combination with PD-L1 blockade in a panel of SCLC models. Results: We demonstrate that inhibition of WEE1 induces G2/M cell cycle arrest, DNA damage, and cytosolic DNA accumulation in SCLC models. We further show that WEE1 targeting activates the STING-TBK1-IRF3 pathway which increases type I interferons (IFN-α and IFN-β) and pro-inflammatory chemokines (CXCL10 and CCL5), facilitating an immune response via CD8+ cytotoxic T-cell infiltration. We further show that WEE1 inhibition concomitantly activates the STAT1 pathway, increasing IFN-γ and PD-L1 expression. Consistent with these findings, combined WEE1 inhibition (AZD1775) and PD-L1 blockade caused remarkable tumor regression, activation of type I and II interferon pathways, and infiltration of cytotoxic T-cells in multiple immunocompetent SCLC genetically engineered mouse models, including an aggressive model with stabilized MYC. Conclusions: Our study demonstrates the cell-autonomous and immune-stimulating activity of WEE1 inhibition in SCLC. Given the increasing importance of immunotherapy for the management of SCLC and that WEE1 inhibitors are already in clinical trials, combining a WEE1 inhibitor with PD-L1 blockade may offer a particularly attractive strategy for the treatment of SCLC and contribute to the rapid translation of this combination into the clinic. Citation Format: Triparna Sen. WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B028.

Macrophage Mediated Abscopal Effects of Radiation Therapy

Radiation therapy (RT) can activate both innate and adaptive immune responses, and the combination of RT and immunotherapy is thought to be a promising strategy. We found that the "don't-eat-me" molecule CD47 is highly expressed on the surface of small cell lung cancer (SCLC) cells, a highly metastatic form of lung cancer, and that blockade of CD47 can enhance the phagocytosis of SCLC cells by macrophages. In this study, we investigated whether combining CD47 blockade and RT could synergize to inhibit the tumor growth of SCLC as well as other cancers in preclinical models. We evaluated efficacy and immune responses by RT and CD47 blockade in SCLC preclinical models as well as in colon cancer and lymphoma preclinical models. We engrafted cancer cells into both flanks of recipient mice and irradiated only one side of tumors with or without CD47 antibodies to investigate local and systemic effects of RT and CD47 blockade. We also used liver metastases models and endogenous lung tumor models to investigate anti-tumor effects and immune responses in their tumor microenvironments. In human patients, we investigated immune responses after RT using CIBERSORTx. We found CD47 blockade potently enhances the local antitumor effects of RT in all the pre-clinical models of SCLC we tested. Strikingly, CD47 blockade also stimulates systemic "abscopal" effects inhibiting non-irradiated SCLC tumors in mice receiving RT. These effects are observed in liver metastases models and endogenous lung tumor models as well as subcutaneous tumor models. Surprisingly, these abscopal effects are independent of T cells but require macrophages that migrate into non-irradiated tumor sites in response to inflammatory signals produced by RT and are locally activated by CD47 blockade to phagocytose cancer cells. Similar abscopal antitumor effects were observed in other cancer models treated with RT and CD47 blockade. We also found that RT increases tumor infiltrating macrophages in human cancer patients. Interestingly, these abscopal antitumor effects are enhanced by PD-1 blockade in some models. Our data clearly demonstrate macrophage-mediated abscopal responses of RT when combined with CD47 blockade in a range of cancer models. The systemic activation of antitumor macrophages following radiation and CD47 blockade may be particularly important in cancer patients who suffer from metastatic disease. RT is a part of standard-of-care and CD47-blocking strategies are in clinical trials, therefore our observations may be rapidly translatable to cancer patients.

P53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism

The p53 tumor suppressor regulates multiple context-dependent tumor suppressive programs. Although p53 is mutated in ~90% of small cell lung cancer (SCLC) tumors, how p53 mediates tumor suppression in this context is unknown. Here, using a mouse model of SCLC in which endogenous p53 expression can be conditionally and temporally regulated, we show that SCLC tumors maintain a requirement for p53 inactivation. However, we identify tumor subtype heterogeneity between SCLC tumors such that p53 reactivation induces senescence in a subset of tumors, while in others, p53 induces necrosis. We pinpoint cyclophilins as critical determinants of a p53-induced transcriptional program that is specific to SCLC tumors and cell lines poised to undergo p53-mediated necrosis. Importantly, inhibition of cyclophilin isomerase activity, or genetic ablation of specific cyclophilin genes, suppresses p53-mediated necrosis by limiting p53 transcriptional output without impacting p53 chromatin binding. Our study demonstrates that intertumoral heterogeneity in SCLC influences the biological response to p53 restoration, describes a cyclophilin-dependent mechanism of p53-regulated cell death, and uncovers putative mechanisms for the treatment of this most-recalcitrant tumor type.

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.

First-in-human study of ABBV-011, a seizure-related homolog protein 6 (SEZ6)–targeting antibody-drug conjugate, in patients with small cell lung cancer.

3002 Background: Small cell lung cancer (SCLC) has a dismal prognosis and new therapies are urgently needed. SEZ6 is a transmembrane protein expressed in SCLC tumors that may be used as a therapeutic target. ABBV-011 is an antibody-drug conjugate (ADC) targeting SEZ6 with a calicheamicin payload, which has shown antitumor activity in preclinical models of SCLC. Preliminary results from the monotherapy dose-escalation and -expansion cohorts of the first-in-human ABBV-011 study are presented. Methods: Phase 1, open-label, multicenter study (NCT03639194) of ABBV-011 alone or in combination with budigalimab, a programmed cell death 1 inhibitor. Primary objectives were to assess the safety and tolerability and to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose of ABBV-011. Adults (≥18 years) with relapsed/refractory SCLC (1–3 lines of prior therapy) were enrolled. Dose escalation was guided by Bayesian continual reassessment method. ABBV-011 was administered intravenously at doses from 0.3 to 2.0 mg/kg once every 3 weeks. Dose expansion was conducted in SEZ6-selected patients. Results: At data cutoff on August 22, 2022, 99 patients were treated with ABBV-011 monotherapy. Median age was 63 years (range, 41–79), 50% of patients were male, and 68% had received ≥2 prior therapies. ABBV-011 ADC pharmacokinetics were approximately dose-proportional with an elimination half-life of 4.6 days across the dose range of 0.3–2.0 mg/kg. In dose escalation (n=26), 1 patient had a dose-limiting toxicity of grade (G) 3 fatigue at 2.0 mg/kg. We report safety and efficacy results for 40 patients in the dose-expansion 1.0-mg/kg ABBV-011 cohort. Median duration of treatment was 12 weeks (range, 1.9–63.3). Treatment-emergent adverse events (TEAEs) occurred in 39 (98%) patients, the most frequent being fatigue (48%), nausea (45%), anorexia (38%), thrombocytopenia (38%), and vomiting (35%). G3 TEAEs occurred in 18 (45%) patients, the most frequent being fatigue, thrombocytopenia, and neutropenia (10% each); 1 G4 TEAE of dyspnea was reported. Seven patients died due to malignant neoplasm/disease progression (n=6) or respiratory distress (n=1); none were related to ABBV-011. Hepatotoxicity was observed, including G≥2 TEAEs of hyperbilirubinemia (18%), increased gamma-glutamyltransferase (8%), ascites (5%), veno-occlusive liver disease (3%), and portal hypertension (3%). Confirmed objective response rate was 25% (10 partial responses [PR]), with median duration of response of 4.2 months (95% CI: 2.6, 6.7). Clinical benefit rate (CBR) was 65% (10 PR and 16 stable disease) and CBR lasting >12 weeks was 43%. The median progression-free survival was 3.5 months. Conclusions: The MTD was not reached and ABBV-011 was well tolerated at 1.0 mg/kg with promising antitumor activity observed. Further evaluation of ABBV-011 is ongoing. Clinical trial information: NCT03639194 .

Single-Cell Evolutionary Analysis Reveals Drivers of Plasticity and Mediators of Chemoresistance in Small Cell Lung Cancer.

Understanding the evolutionary dynamics of SCLC can shed light on key mechanisms such as cellular plasticity, heterogeneity, and chemoresistance.

IL-18-secreting CAR T cells targeting DLL3 are highly effective in small cell lung cancer models.

Patients with small cell lung cancer (SCLC) generally have a poor prognosis and a median overall survival of only about 13 months, indicating the urgent need for novel therapies. Delta-like protein 3 (DLL3) has been identified as a tumor-specific cell surface marker on neuroendocrine cancers, including SCLC. In this study, we developed a chimeric antigen receptor (CAR) against DLL3 that displays antitumor efficacy in xenograft and murine SCLC models. CAR T cell expression of the proinflammatory cytokine IL-18 greatly enhanced the potency of DLL3-targeting CAR T cell therapy. In a murine metastatic SCLC model, IL-18 production increased the activation of both CAR T cells and endogenous tumor-infiltrating lymphocytes. We also observed an increased infiltration, repolarization, and activation of antigen-presenting cells. Additionally, human IL-18-secreting anti-DLL3 CAR T cells showed an increased memory phenotype, less exhaustion, and induced durable responses in multiple SCLC models, an effect that could be further enhanced with anti-PD-1 blockade. All together, these results define DLL3-targeting CAR T cells that produce IL-18 as a potentially promising novel strategy against DLL3-expressing solid tumors.