Functional Characterisation of the ATOH1 Molecular Subtype Indicates a Pro-Metastatic Role in Small Cell Lung Cancer.

Molecular subtypes of Small Cell Lung Cancer (SCLC) have been described based on differential expression of transcription factors (TFs) ASCL1, NEUROD1, POU2F3 and immune-related genes. We previously reported an additional subtype based on expression of the neurogenic TF ATOH1 within our SCLC Circulating tumour cell-Derived eXplant (CDX) model biobank. Here we show that ATOH1 protein was detected in 7/81 preclinical models and 16/102 clinical samples of SCLC. In CDX models, ATOH1 directly regulated neurogenesis and differentiation programs consistent with roles in normal tissues. In ex vivo cultures of ATOH1-positive CDX, ATOH1 was required for cell survival. In vivo, ATOH1 depletion slowed tumour growth and suppressed liver metastasis. Our data validate ATOH1 as a bona fide oncogenic driver of SCLC with tumour cell survival and pro-metastatic functions. Further investigation to explore ATOH1 driven vulnerabilities for targeted treatment with predictive biomarkers is warranted.

Soluble guanylate cyclase signalling mediates etoposide resistance in progressing small cell lung cancer.

Small cell lung cancer (SCLC) has a 5-year survival rate of <7%. Rapid emergence of acquired resistance to standard platinum-etoposide chemotherapy is common and improved therapies are required for this recalcitrant tumour. We exploit six paired pre-treatment and post-chemotherapy circulating tumour cell patient-derived explant (CDX) models from donors with extensive stage SCLC to investigate changes at disease progression after chemotherapy. Soluble guanylate cyclase (sGC) is recurrently upregulated in post-chemotherapy progression CDX models, which correlates with acquired chemoresistance. Expression and activation of sGC is regulated by Notch and nitric oxide (NO) signalling with downstream activation of protein kinase G. Genetic targeting of sGC or pharmacological inhibition of NO synthase re-sensitizes a chemoresistant CDX progression model in vivo, revealing this pathway as a mediator of chemoresistance and potential vulnerability of relapsed SCLC.

The Rare YAP1 Subtype of SCLC Revisited in a Biobank of 39 Circulating Tumor Cell Patient Derived Explant Models: A Brief Report.

INTRODUCTION: Recent consensus defines four SCLC subtypes on the basis of transcription factor expression: ASCL1, NEUROD1, POU2F3, and YAP1. The rare YAP1 subtype is associated with "neuroendocrine (NE)-low" cells among SCLC cell lines and patient samples. We evaluated YAP1 in 39 patients with phenotypically diverse circulating tumor cell-derived explant (CDX) models and revisited YAP1 in terms of prevalence, cell phenotype, and intertumor and intratumor heterogeneity. METHODS: YAP1 transcript and protein expression were assessed by RNA sequencing and immunohistochemistry or multiplexed immunofluorescence of NE and non-NE CDX subpopulations. Physically separated NE and non-NE CDX ex vivo culture lysates were Western blotted for YAP1, NE marker SYP, and AXL. RESULTS: RNA sequencing normalized for the four subtype transcription factors identified YAP1 expression in 14 of 39 CDX. A total of 10 CDX expressed YAP1 protein, and eight had strong YAP1 expression confined to rare non-NE cell clusters. This was confirmed in ex vivo CDX cultures in which adherent non-NE cells lacking SYP expression expressed YAP1. However, in two CDX, weaker cellular YAP1 expression was observed, widely dispersed in SYP-positive NE cells. CONCLUSIONS: YAP1 was predominantly expressed in non-NE cell clusters in SCLC CDX, but two of 39 CDX expressed YAP1 in NE cells. CDX22P, with relatively high YAP1 expression, is an ASCL1 NE subtype with a low NE score and an outlier within this subtype in our CDX biobank. These descriptive data reveal subtly different YAP1 expression profiles, paving the way for functional studies to compare YAP1 signaling in non-NE and low NE cell contexts for potentially personalized therapeutic approaches.

A biobank of small cell lung cancer CDX models elucidates inter- and intratumoral phenotypic heterogeneity.

Although small cell lung cancer (SCLC) is treated as a homogeneous disease, biopsies and preclinical models reveal heterogeneity in transcriptomes and morphology. SCLC subtypes were recently defined by neuroendocrine transcription factor (NETF) expression. Circulating-tumor-cell-derived explant models (CDX) recapitulate donor patients' tumor morphology, diagnostic NE marker expression and chemotherapy responses. We describe a biobank of 38 CDX models, including six CDX pairs generated pretreatment and at disease progression revealing complex intra- and intertumoral heterogeneity. Transcriptomic analysis confirmed three of four previously described subtypes based on ASCL1, NEUROD1 and POU2F3 expression and identified a previously unreported subtype based on another NETF, ATOH1. We document evolution during disease progression exemplified by altered MYC and NOTCH gene expression, increased 'variant' cell morphology, and metastasis without strong evidence of epithelial to mesenchymal transition. This CDX biobank provides a research resource to facilitate SCLC personalized medicine.

Pre-clinical pharmacology of AZD3965, a selective inhibitor of MCT1: DLBCL, NHL and Burkitt's lymphoma anti-tumor activity.

Tumors frequently display a glycolytic phenotype with increased flux through glycolysis and concomitant synthesis of lactate. To maintain glycolytic flux and prevent intracellular acidification, tumors efflux lactate via lactate transporters (MCT1-4). Inhibitors of lactate transport have the potential to inhibit glycolysis and tumor growth. We developed a small molecule inhibitor of MCT1 (AZD3965) and assessed its activity across a panel of cell lines. We explored its antitumor activity as monotherapy and in combination with doxorubicin or rituximab. AZD3965 is a potent inhibitor of MCT1 with activity against MCT2 but selectivity over MCT3 and MCT4. In vitro, AZD3965 inhibited the growth of a range of cell lines especially haematological cells. Inhibition of MCT1 by AZD3965 inhibited lactate efflux and resulted in accumulation of glycolytic intermediates. In vivo, AZD3965 caused lactate accumulation in the Raji Burkitt's lymphoma model and significant tumor growth inhibition. Moreover, AZD3965 can be combined with doxorubicin or rituximab, components of the R-CHOP standard-of-care in DLBCL and Burkitt's lymphoma. Finally, combining lactate transport inhibition by AZD3965 with GLS1 inhibition in vitro, enhanced cell growth inhibition and cell death compared to monotherapy treatment. The ability to combine AZD3965 with novel, and standard-of-care inhibitors offers novel combination opportunities in haematological cancers.

Targeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense oligonucleotide inhibitor of KRAS.

Activating mutations in KRAS underlie the pathogenesis of up to 20% of human tumors, and KRAS is one of the most frequently mutated genes in cancer. Developing therapeutics to block KRAS activity has proven difficult, and no direct inhibitor of KRAS function has entered clinical trials. We describe the preclinical evaluation of AZD4785, a high-affinity constrained ethyl-containing therapeutic antisense oligonucleotide (ASO) targeting KRAS mRNA. AZD4785 potently and selectively depleted cellular KRAS mRNA and protein, resulting in inhibition of downstream effector pathways and antiproliferative effects selectively in KRAS mutant cells. AZD4785-mediated depletion of KRAS was not associated with feedback activation of the mitogen-activated protein kinase (MAPK) pathway, which is seen with RAS-MAPK pathway inhibitors. Systemic delivery of AZD4785 to mice bearing KRAS mutant non-small cell lung cancer cell line xenografts or patient-derived xenografts resulted in inhibition of KRAS expression in tumors and antitumor activity. The safety of this approach was demonstrated in mice and monkeys with KRAS ASOs that produced robust target knockdown in a broad set of tissues without any adverse effects. Together, these data suggest that AZD4785 is an attractive therapeutic for the treatment of KRAS-driven human cancers and warrants further development.