Killing SCLC: insights into how to target a shapeshifting tumor.

Small cell lung cancer (SCLC) is a rapidly growing, highly metastatic, and relatively immune-cold lung cancer subtype. Historically viewed in the laboratory and clinic as a single disease, new discoveries suggest that SCLC comprises multiple molecular subsets. Expression of MYC family members and lineage-related transcription factors ASCL1, NEUROD1, and POU2F3 (and, in some studies, YAP1) define unique molecular states that have been associated with distinct responses to a variety of therapies. However, SCLC tumors exhibit a high degree of intratumoral heterogeneity, with recent studies suggesting the existence of tumor cell plasticity and phenotypic switching between subtype states. While SCLC plasticity is correlated with, and likely drives, therapeutic resistance, the mechanisms underlying this plasticity are still largely unknown. Subtype states are also associated with immune-related gene expression, which likely impacts response to immune checkpoint blockade and may reveal novel targets for alternative immunotherapeutic approaches. In this review, we synthesize recent discoveries on the mechanisms of SCLC plasticity and how these processes may impinge on antitumor immunity.

Paracrine signaling between tumor subclones of mouse SCLC: a critical role of ETS transcription factor Pea3 in facilitating metastasis.

Tumor heterogeneity can create a unique symbiotic tumor microenvironment. Earlier, we showed that clonal evolution in mouse small cell lung cancer (SCLC) can result in subclones that, upon cografting, endow the neuroendocrine tumor cells with metastatic potential. We now show that paracrine signaling between SCLC subclones is a critical requirement in the early steps of the metastatic process, such as local invasion and intravasation. We further show evidence that paracrine signaling via fibroblast growth factor 2 (Fgf2) and Mapk between these diverged tumor subclones causes enhanced expression of the Pea3 (polyomavirus enhancer activator 3) transcription factor, resulting in metastatic dissemination of the neuroendocrine tumor subclones. Our data reveal for the first time paracrine signaling between tumor cell subclones in SCLC that results in metastatic spread of SCLC.

Delineating the roles of Grhl2 in craniofacial development through tissue-specific conditional deletion and epistasis approaches in mouse.

BACKGROUND: The highly conserved Grainyhead-like (Grhl) family of transcription factors play critical roles in the development of the neural tube and craniofacial skeleton. In particular, deletion of family member Grainyhead-like 2 (Grhl2) leads to mid-gestational embryonic lethality, maxillary clefting, abdominoschisis, and both cranial and caudal neural tube closure defects. These highly pleiotropic and systemic defects suggest that Grhl2 plays numerous critical developmental roles to ensure correct morphogenesis and patterning. RESULTS: Here, using four separate Cre-lox conditional deletion models, as well as one genetic epistasis approach (Grhl2+/- ;Edn1+/- double heterozygous mice) we have investigated tissue-specific roles of Grhl2 in embryonic development, with a particular focus on the craniofacial skeleton. We find that loss of Grhl2 in the pharyngeal epithelium (using the ShhCre driver) leads to low-penetrance micrognathia, whereas deletion of Grhl2 within the ectoderm of the pharynx (NestinCre ) leads to small, albeit significant, differences in the proximal-distal elongation of both the maxilla and mandible. Loss of Grhl2 in endoderm (Sox17-2aiCre ) resulted in noticeable lung defects and a single instance of secondary palatal clefting, although formation of other endoderm-derived organs such as the stomach, bladder and intestines was not affected. Lastly, deletion of Grhl2 in cells of the neural crest (Wnt1Cre ) did not lead to any discernible defects in craniofacial development, and similarly, our epistasis approach did not detect any phenotypic consequences of loss of a single allele of both Grhl2 and Edn1. CONCLUSION: Taken together, our study identifies a pharyngeal-epithelium intrinsic, non-cell-autonomous role for Grhl2 in the patterning and formation of the craniofacial skeleton, as well as an endoderm-specific role for Grhl2 in the formation and establishment of the mammalian lung.

Critical cancer vulnerabilities identified by unbiased CRISPR/Cas9 screens inform on efficient cancer Immunotherapy.

The mutational landscape of human cancers is highly complex. While next generation sequencing aims to comprehensively catalogue somatic alterations in tumor cells, it fails to delineate driver from passenger mutations. Functional genomic approaches, particularly CRISPR/Cas9, enable both gene discovery, and annotation of gene function. Indeed, recent CRISPR/Cas9 technologies have flourished with the development of more sophisticated and versatile platforms capable of gene knockouts to high throughput genome wide editing of a single nucleotide base. With new platforms constantly emerging, it can be challenging to navigate what CRISPR tools are available and how they can be effectively applied to understand cancer biology. This review provides an overview of current and emerging CRISPR technologies and their power to model cancer and identify novel treatments. Specifically, how CRISPR screening approaches have been exploited to enhance immunotherapies through the identification of tumor intrinsic and extrinsic mechanisms to escape immune recognition will be discussed.

Lung Basal Stem Cells Rapidly Repair DNA Damage Using the Error-Prone Nonhomologous End-Joining Pathway.

Lung squamous cell carcinoma (SqCC), the second most common subtype of lung cancer, is strongly associated with tobacco smoking and exhibits genomic instability. The cellular origins and molecular processes that contribute to SqCC formation are largely unexplored. Here we show that human basal stem cells (BSCs) isolated from heavy smokers proliferate extensively, whereas their alveolar progenitor cell counterparts have limited colony-forming capacity. We demonstrate that this difference arises in part because of the ability of BSCs to repair their DNA more efficiently than alveolar cells following ionizing radiation or chemical-induced DNA damage. Analysis of mice harbouring a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in DNA damage repair by nonhomologous end joining (NHEJ), indicated that BSCs preferentially repair their DNA by this error-prone process. Interestingly, polyploidy, a phenomenon associated with genetically unstable cells, was only observed in the human BSC subset. Expression signature analysis indicated that BSCs are the likely cells of origin of human SqCC and that high levels of NHEJ genes in SqCC are correlated with increasing genomic instability. Hence, our results favour a model in which heavy smoking promotes proliferation of BSCs, and their predilection for error-prone NHEJ could lead to the high mutagenic burden that culminates in SqCC. Targeting DNA repair processes may therefore have a role in the prevention and therapy of SqCC.

Lung morphogenesis is orchestrated through Grainyhead-like 2 (Grhl2) transcriptional programs.

The highly conserved transcription factor Grainyhead-like 2 (Grhl2) exhibits a dynamic expression pattern in lung epithelium throughout embryonic development. Using a conditional gene targeting approach to delete Grhl2 in the developing lung epithelium, our results demonstrate that Grhl2 plays multiple roles in lung morphogenesis that are essential for respiratory function. Loss of Grhl2 leads to impaired ciliated cell differentiation and perturbed formation of terminal saccules. Critically, a substantial increase in Sox9-positive distal tip progenitor cells was observed following loss of Grhl2, suggesting that Grhl2 plays an important role in branching morphogenesis. Gene transcription profiling of Grhl2-deficient lung epithelial cells revealed a significant down regulation of Elf5, a member of the Ets family of transcription factors. Furthermore, ChIP and comparative genomic analyzes confirmed that Elf5 is a direct transcriptional target of Grhl2. Taken together, these results support the hypothesis that Grhl2 controls normal lung morphogenesis by tightly regulating the activity of distal tip progenitor cells.

Multiple cells-of-origin of mutant K-Ras-induced mouse lung adenocarcinoma.

Much controversy surrounds the cell-of-origin of mutant K-Ras (K-RasG12D)-induced lung adenocarcinoma. To shed light on this issue, we have used technology that enables us to conditionally target K-RasG12D expression in Surfactant Protein C (SPC)(+) alveolar type 2 cells and in Clara cell antigen 10 (CC10)(+) Clara cells by use of cell-type-restricted recombinant Adeno-Cre viruses. Experiments were performed both in the presence and absence of the tumor suppressor gene p53, enabling us to assess what effect the cell-of-origin and the introduced genetic lesions have on the phenotypic characteristics of the resulting adenocarcinomas. We conclude that both SPC-expressing alveolar type 2 cells and CC10-expressing Clara cells have the ability to initiate malignant transformation following the introduction of these genetic alterations. The lungs of K-Ras(lox-Stop-lox-G12D/+) and K-Ras(lox-Stop-lox-G12D/+);tumor suppressor gene Trp53(F/F) mice infected with Adeno5-SPC-Cre and Adeno5-CC10-Cre viruses displayed differences in their tumor spectrum, indicating distinct cellular routes of tumor initiation. Moreover, using a multicolor Cre reporter line, we demonstrate that the resulting tumors arise from a clonal expansion of switched cells. Taken together, these results indicate that there are multiple cellular paths to K-RasG12D-induced adenocarcinoma and that the initiating cell influences the histopathological phenotype of the tumors that arise.

The LIM-domain only protein 4 contributes to lung epithelial cell proliferation but is not essential for tumor progression.

BACKGROUND: The lung is constantly exposed to environmental challenges and must rapidly respond to external insults. Mechanisms involved in the repair of the damaged lung involve expansion of different epithelial cells to repopulate the injured cellular compartment. However, factors regulating cell proliferation following lung injury remain poorly understood. Here we studied the role of the transcriptional regulator Lmo4 during lung development, in the regulation of adult lung epithelial cell proliferation following lung damage and in the context of oncogenic transformation. METHODS: To study the role of Lmo4 in embryonic lung development, lung repair and tumorigenesis, we used conditional knock-out mice to delete Lmo4 in lung epithelial cells from the first stages of lung development. The role of Lmo4 in lung repair was evaluated using two experimental models of lung damage involving chemical and viral injury. The role of Lmo4 in lung tumorigenesis was measured using a mouse model of lung adenocarcinoma in which the oncogenic K-Ras protein has been knocked into the K-Ras locus. Overall survival difference between genotypes was tested by log rank test. Difference between means was tested using one-way ANOVA after assuring that assumptions of normality and equality of variance were satisfied. RESULTS: We found that Lmo4 was not required for normal embryonic lung morphogenesis. In the adult lung, loss of Lmo4 reduced epithelial cell proliferation and delayed repair of the lung following naphthalene or flu-mediated injury, suggesting that Lmo4 participates in the regulation of epithelial cell expansion in response to cellular damage. In the context of K-Ras(G12D)-driven lung tumor formation, Lmo4 loss did not alter overall survival but delayed initiation of lung hyperplasia in K-Ras(G12D) mice sensitized by naphthalene injury. Finally, we evaluated the expression of LMO4 in tissue microarrays of early stage non-small cell lung cancer and observed that LMO4 is more highly expressed in lung squamous cell carcinoma compared to adenocarcinoma. CONCLUSIONS: Together these results show that the transcriptional regulator Lmo4 participates in the regulation of lung epithelial cell proliferation in the context of injury and oncogenic transformation but that Lmo4 depletion is not sufficient to prevent lung repair or tumour formation.

Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation.

The transcription factor Gata-3 is a defining marker of the 'luminal' subtypes of breast cancer. To gain insight into the role of Gata-3 in breast epithelial development and oncogenesis, we have explored its normal function within the mammary gland by conditionally deleting Gata-3 at different stages of development. We report that Gata-3 has essential roles in the morphogenesis of the mammary gland in both the embryo and adult. Through the discovery of a novel marker (beta3-integrin) of luminal progenitor cells and their purification, we demonstrate that Gata-3 deficiency leads to an expansion of luminal progenitors and a concomitant block in differentiation. Remarkably, introduction of Gata-3 into a stem cell-enriched population induced maturation along the alveolar luminal lineage. These studies provide evidence for the existence of an epithelial hierarchy within the mammary gland and establish Gata-3 as a critical regulator of luminal differentiation.

A Multiplexed Approach to Assess Small Cell Lung Cancer Subtype Heterogeneity in Primary and Patient-Derived Tumor Samples.

Unlocking the heterogeneity of cancers is crucial for developing therapeutic approaches that effectively eradicate disease. As our understanding of markers specific to cancer subclones or subtypes expands, there is a growing demand for advanced technologies that enable the simultaneous investigation of multiple targets within an individual tumor sample. Indeed, multiplex approaches offer distinct benefits, particularly when tumor specimens are small and scarce. Here we describe the utility of two fluorescence-based multiplex approaches; fluorescent Western blots, and multiplex immunohistochemistry (Opal™) staining to interrogate heterogeneity, using small cell lung cancer as an example. Critically, the coupling of Opal™ staining with advanced image quantitation, permits the dissection of cancer cell phenotypes at a single cell level. These approaches can be applied to patient biopsies and/or patient-derived xenograft (PDX) models and serve as powerful methodologies for assessing tumor cell heterogeneity in response to therapy or between metastatic lesions across diverse tissue sites.

Molecular and Pathologic Characterization of YAP1-Expressing Small Cell Lung Cancer Cell Lines Leads to Reclassification as SMARCA4-Deficient Malignancies.

PURPOSE: The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial. EXPERIMENTAL DESIGN: To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue. RESULTS: Unexpectedly, pathogenic mutations in SMARCA4 were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile SMARCA4-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non-small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype. CONCLUSIONS: SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC. See related commentary by Rekhtman, p. 1708.

Next batter up! Targeting cancers with KRAS-G12D mutations.

KRAS is the most frequently mutated oncogene in cancer. Activating mutations in codon 12, especially G12D, have the highest prevalence across a range of carcinomas and adenocarcinomas. With inhibitors to KRAS-G12D now entering clinical trials, understanding the biology of KRAS-G12D cancers, and identifying biomarkers that predict therapeutic response is crucial. In this Review, we discuss the genomics and biology of KRAS-G12D adenocarcinomas, including histological features, transcriptional landscape, the immune microenvironment, and how these factors influence response to therapy. Moreover, we explore potential therapeutic strategies using novel G12D inhibitors, leveraging knowledge gained from clinical trials using G12C inhibitors.

On Target: An Intrapulmonary Transplantation Method for Modelling Lung Tumor Development in its Native Microenvironment.

The development of in vivo lung cancer models that faithfully mimic the human disease is a crucial research tool for understanding the molecular mechanisms driving tumorigenesis. Subcutaneous transplantation assays are commonly employed, likely due to their amenability to easily monitor tumor growth and the simplistic nature of the technique to deliver tumor cells. Importantly however, subcutaneous tumors grow in a microenvironment that differs from that resident within the lung. To circumvent this limitation, here we describe the development of an intrapulmonary (iPUL) orthotopic transplantation method that enables the delivery of lung cancer cells, with precision, to the left lung lobe of recipient mice. Critically, this allows for the growth of lung cancer cells within their native microenvironment. The coupling of iPUL transplantation with position emission tomography (PET) imaging permits the serial detection of tumors in vivo and serves as a powerful tool to trace lung tumor growth and dissemination over time in mouse disease models.

Design and characterization of a heterobifunctional degrader of KEAP1.

The Kelch-like ECH-associated protein 1 (KEAP1) - nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway senses reactive oxygen species and regulates cellular oxidative stress. Inhibiting KEAP1 to activate the NRF2 antioxidant response has been proposed as a promising strategy to treat chronic diseases caused by oxidative stress. Here, we developed a proteolysis targeting chimera (PROTAC) that depletes KEAP1 from cells through the ubiquitin-proteasome pathway. A previously developed KEAP1 inhibitor and thalidomide were incorporated in the heterobifunctional design of the PROTAC as ligands for KEAP1 and CRBN recruitment, respectively. Optimization of the chemical composition and linker length resulted in PROTAC 14 which exhibited potent KEAP1 degradation with low nanomolar DC50 in HEK293T (11 nM) and BEAS-2B (<1 nM) cell lines. Furthermore, PROTAC 14 increased the expression of NRF2 regulated antioxidant proteins and prevented cell death induced by reactive oxygen species. Together, these results established a blueprint for further development of KEAP1-targeted heterobifunctional degraders and will facilitate the study of the biological consequences of KEAP1 removal from cells. This approach represents an alternative therapeutic strategy to existing treatments for diseases caused by oxidative stress.

Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes.

Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR-Cas9 screens, we identify specific roles for MTF2-PRC2.1, PCGF1-PRC1.1 and Menin-KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin-KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.

Early immune pressure initiated by tissue-resident memory T cells sculpts tumor evolution in non-small cell lung cancer.

Tissue-resident memory T (TRM) cells provide immune defense against local infection and can inhibit cancer progression. However, it is unclear to what extent chronic inflammation impacts TRM activation and whether TRM cells existing in tissues before tumor onset influence cancer evolution in humans. We performed deep profiling of healthy lungs and lung cancers in never-smokers (NSs) and ever-smokers (ESs), finding evidence of enhanced immunosurveillance by cells with a TRM-like phenotype in ES lungs. In preclinical models, tumor-specific or bystander TRM-like cells present prior to tumor onset boosted immune cell recruitment, causing tumor immune evasion through loss of MHC class I protein expression and resistance to immune checkpoint inhibitors. In humans, only tumors arising in ES patients underwent clonal immune evasion, unrelated to tobacco-associated mutagenic signatures or oncogenic drivers. These data demonstrate that enhanced TRM-like activity prior to tumor development shapes the evolution of tumor immunogenicity and can impact immunotherapy outcomes.

Absence of pro-survival A1 has no impact on inflammatory cell survival in vivo during acute lung inflammation and peritonitis.

Inflammation is a natural defence mechanism of the body to protect against pathogens. It is induced by immune cells, such as macrophages and neutrophils, which are rapidly recruited to the site of infection, mediating host defence. The processes for eliminating inflammatory cells after pathogen clearance are critical in preventing sustained inflammation, which can instigate diverse pathologies. During chronic inflammation, the excessive and uncontrollable activity of the immune system can cause extensive tissue damage. New therapies aimed at preventing this over-activity of the immune system could have major clinical benefits. Here, we investigated the role of the pro-survival Bcl-2 family member A1 in the survival of inflammatory cells under normal and inflammatory conditions using murine models of lung and peritoneal inflammation. Despite the robust upregulation of A1 protein levels in wild-type cells upon induction of inflammation, the survival of inflammatory cells was not impacted in A1-deficient mice compared to wild-type controls. These findings indicate that A1 does not play a major role in immune cell homoeostasis during inflammation and therefore does not constitute an attractive therapeutic target for such morbidities.

Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer.

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.

Inhibition of LSD1 with Bomedemstat Sensitizes Small Cell Lung Cancer to Immune Checkpoint Blockade and T-Cell Killing.

PURPOSE: The addition of immune checkpoint blockade (ICB) to platinum/etoposide chemotherapy changed the standard of care for small cell lung cancer (SCLC) treatment. However, ICB addition only modestly improved clinical outcomes, likely reflecting the high prevalence of an immunologically "cold" tumor microenvironment in SCLC, despite high mutational burden. Nevertheless, some patients clearly benefit from ICB and recent reports have associated clinical responses to ICB in SCLC with (i) decreased neuroendocrine characteristics and (ii) activation of NOTCH signaling. We previously showed that inhibition of the lysine-specific demethylase 1a (LSD1) demethylase activates NOTCH and suppresses neuroendocrine features of SCLC, leading us to investigate whether LSD1 inhibition would enhance the response to PD-1 inhibition in SCLC. EXPERIMENTAL DESIGN: We employed a syngeneic immunocompetent model of SCLC, derived from a genetically engineered mouse model harboring Rb1/Trp53 inactivation, to investigate combining the LSD1 inhibitor bomedemstat with anti-PD-1 therapy. In vivo experiments were complemented by cell-based studies in murine and human models. RESULTS: Bomedemstat potentiated responses to PD-1 inhibition in a syngeneic model of SCLC, resulting in increased CD8+ T-cell infiltration and strong tumor growth inhibition. Bomedemstat increased MHC class I expression in mouse SCLC tumor cells in vivo and augmented MHC-I induction by IFNγ and increased killing by tumor-specific T cells in cell culture. CONCLUSIONS: LSD1 inhibition increased MHC-I expression and enhanced responses to PD-1 inhibition in vivo, supporting a new clinical trial to combine bomedemstat with standard-of-care PD-1 axis inhibition in SCLC.

Exploring natural killer cell immunology as a therapeutic strategy in lung cancer.

Cytotoxic immune cells are key in the control of tumor development and progression. Natural killer (NK) cells are the cytotoxic arm of the innate immune system with the capability to kill tumor cells and surveil tumor cell dissemination. As such, the interest in harnessing NK cells in tumor control is increasing in many solid tumor types, including lung cancer. Here, we review the pre-clinical models used to unveil the role of NK cells in immunosurveillance of solid tumors and highlight measures to enhance NK cell activity. Importantly, the development of NK immunotherapy is rapidly evolving. Enhancing the NK cell response can be achieved using two broad modalities: enhancing endogenous NK cell activity, or performing adoptive transfer of pre-activated NK cells to patients. Numerous clinical trials are evaluating the efficacy of NK cell immunotherapy in isolation or in combination with standard treatments, with encouraging initial results. Pre-clinical studies and early phase clinical trials suggest that patients with solid tumors, including lung cancer, have the potential to benefit from recent developments in NK cell immunotherapy.