Small cell lung carcinoma (SCLC) is a highly lethal, metastasis-prone solid tumor that is difficult to treat with standard therapies. While SCLC is often initially responsive to combination chemotherapies, chemoresistance rapidly emerges in advanced cancer and leads to a 5-year survival rate of 5-6%. Additionally, unlike cancers like melanoma, the combination of immune checkpoint inhibition with standard chemotherapy only modestly improves patient outcomes. The lethality and adaptability of SCLC makes it especially important to identify novel therapies to treat patients with these cancers – a challenge the MacPherson Lab, Human Biology Division, answered by uncovering a new link between SCLC proliferation and a specific protein regulation pathway. Their paper is published in Genes and Development.
To identify targetable genes essential for SCLC growth and survival, the authors began with an exploratory, genome wide CRISPR/Cas9 screen. A genome wide CRISPR/Cas9 screen is an unbiased tool that aims to identify genes involved in a particular process by individually “breaking” every gene in the genome and observing which genes have an effect. The MacPherson Lab conducted their screen in five independent cell lines derived from a mouse model of SCLC (mSCLC) and three isolates of immortalized mouse embryonic fibroblasts (MEFs), a non-cancerous population, for comparison. The goal was to locate novel, druggable genes that were depleted in the mSCLC cells relative to the MEFs. While their screen uncovered a few well-characterized SCLC tumor suppressors, such as PTEN and CREBBP, “our screen results [also] uncovered numerous members of the neddylation pathway”, said researcher Dr. Justin Norton.
Neddylation refers to the addition of a small protein, NEDD8 (neuronal precursor cell-expressed developmentally downregulated protein 8), to lysine residues in target proteins. The main targets of neddylation are cullin proteins, which require this modification for their function as scaffold proteins that facilitate the ubiquitylation and degradation of various substrates. “The existence of a novel neddylation inhibitor in early-stage clinical trials further intrigued us to this pathway,” said Dr. Norton. MLN4924 (Pevonedistat) is a small molecule that inhibits neddylation activation enzyme 1 (NAE1), preventing neddylation and causing cell cycle arrest, apoptosis, senescence, and autophagy in many cancer types. Its status in clinical trials to treat both solid and liquid cancers made this drug a prime candidate to evaluate for SCLC treatment.
The MacPherson Lab hypothesized that MLN4924 could be an effective and specific treatment for SCLC. They first used the drug on two mSCLC cell lines, two MEF isolates, and two cell lines from mouse models of non-small cell lung cancer (NSCLC). The drug had a potent effect on the mSCLC cell lines’ survival at a much lower dose (IC50 between 0.4-1.2µM) when compared to MEF and NSCLC lines (IC50 between 3-5µM and 10-15µM, respectively). A Western blot (WB) confirmed the loss of neddylated cullin proteins in the mSCLC cell lines following 48h of MLN4924 treatment, which meant the drug was working on-target.
Encouraged by these results, the authors moved on to assess the therapeutic potential of MLN4924 on human SCLC in a panel of patient-derived xenograph (PDX) models. To create a PDX model, researchers transplant cells derived from human patient tumors into immunodeficient mice and evaluate tumor growth in various conditions. They sourced cells from multiple tumor types, including both a human SCLC and an extrapulmonary small cell carcinoma from a patient’s cervix. Tumor cells in mice treated with MLN4924 were stagnant and showed virtually no growth, and further examination on a cellular level showed markedly decreased markers of cell proliferation and increased markers of cell death. “Neddylation inhibition dramatically slowed growth in both cultured cell lines and in vivo PDX models of small cell carcinoma,” said Dr. Norton. “Excitingly, this coincided with a broad and robust transcriptional downregulation of neuroendocrine genes, for which expression is necessary in many subsets of SCLC.” Overall, it appeared the drug was working to slow carcinoma growth, and the downregulation of neuroendocrine genes pointed to a potential mechanism.
Both SCLC and extrapulmonary SCC characteristically express multiple neuroendocrine markers, including master regulators ASCL1, INSM1, and FOXA2. RNAseq revealed that these master regulators and their targets were downregulated in MLN4924-treated PDX models, and protein levels were also significantly lower. Knockdown of two neddylation pathway members, NEDD8 and RBX1, by short hairpin RNA (shRNA) mimicked the results of MLN4924 treatment and was coupled with strongly reduced proliferation. “This work is the first to link the neddylation pathway as a regulator of the neuroendocrine cell state, a defining feature of small cell carcinoma” said Dr. Norton, highlighting the innovation of this study.
As SCLC frequently acquires resistance to therapies, the MacPherson Lab concluded their project by exploring how SCLC tumors might escape treatment with MLN4924 in the future. Using the same CRISPR/Cas9 genome wide screen approach as before, they found that members of the COP9 signalosome, which removes NEDD8 from and deactivates cullin-containing complexes, could counteract the MLN4924 treatment. Specifically, knockout of the COP9 subunit COPS4 increased neddylation of cullins, partially restored protein levels of the master neuroendocrine regulators, and lowered apoptotic markers cleaved CASP7 and cleaved PARP. These experiments demonstrated that loss of COPS4 is a strong mechanism of resistance to MLN4924 treatment that might be of concern in future clinical applications.
This research exposed many exciting leads to follow. The MacPherson Lab would like to explore whether the sensitivity of the extrapulmonary SCC to MLN4924 extends to multiple sites-of-origin for high-grade neuroendocrine tumors, for instance. Additionally, while the PDX data presented in this study suggest that MLN4924 can be powerful on its own, the drug might have even stronger or more broad effects if used in conjunction with other chemotherapies or immune checkpoint inhibitors. Answering such complicated questions requires complicated methods, and the MacPherson Lab is thankful for the resources afforded to the lab by their membership in the Cancer Consortium. “Shared Resources supported by the Fred Hutch/UW Cancer Consortium, including Genomics and Bioinformatics, Comparative Medicine, and Experimental Histopathology were all critical for the success of this project,” said Dr. MacPherson. The discovery of a potential novel therapeutic is an exciting development for a diagnosis as high-stakes as small cell lung carcinoma.
This work was supported by National Institutes of Health, Lung Cancer Research Foundation/Uniting Against Lung Cancer, a National Cancer Institute Cancer Center support grant, the Specialized Program of Research Excellence (SPORE) in lung cancer, and the Evergreen Fund Pilot Award.
Cancer Consortium faculty member Dr. David MacPherson and the Shared Resources at the Fred Hutchinson Cancer Research Center contributed to this research.
Norton JP, Augert A, Eastwood E, Basom R, Rudin CM, MacPherson D. Protein neddylation as a therapeutic target in pulmonary and extrapulmonary small cell carcinomas. Genes & development. 2021 Jun 1;35(11-12):870-87. doi:10.1101/gad.348316.121