Overview: Small cell lung cancer (SCLC) is a recalcitrant cancer with no approved targeted therapies. This is partly because SCLC is driven by loss of function (LOF) mutations in tumor suppressor genes and lacks mutations in druggable oncogenes. Whole exome sequencing studies have demonstrated that SCLCs harbor near universal LOF mutations in the tumor suppressor genes RB1 and TP53, and ~25% of SCLCs have mutually exclusive LOF mutations in NOTCH. Recently it has been appreciated that SCLC consists of at least four molecular subtypes which include the ASCL1, NEUROD1, POU2F3, and inflammatory subtypes. ASCL1, NEUROD1, and POU2F3 themselves are transcription factors and are selective dependencies in each of their respective subtypes suggesting that if "druggable" they could be good make good therapeutic targets. Our laboratory uses innovative functional genomic approaches including CRISPR/Cas9 screening to uncover regulation of these transcription factor drivers with the goal to identify "druggable" mechanisms that ultimately block their function. We also use in vivo CRISPR-based mouse modeling to understand how epigenetic drivers control these transcription factor subtypes and the consequences of inhibiting epigenetic drivers on subtype identity, plasticity, and tumor immunogenicity.
Identification of Mechanisms that Control SCLC Transcription Factor Subtypes: Our previous work and work from other laboratories have shown that multiple epigenetic modifying enzymes including LSD1, EZH2, and KDM5A function to control expression of neuroendocrine transcription factors in SCLC. For example, the histone demethylases KDM5A and LSD1 can function to repress NOTCH, which is required to sustain ASCL1 expression in SCLC. ASCL1 is the dominant SCLC subtype and is a dependency in many of these SCLCs and required for SCLC tumorigenesis in mouse models. Our laboratory is interested in understanding mechanisms that SCLCs utilize to sustain high expression of neuroendocrine transcription factors, such as ASCL1. We utilize novel unbiased CRISPR/Cas9 positive selection screening approaches to interrogate how SCLC neuroendocrine transcription factors are regulated in SCLC with goal being to identify new therapeutic strategies to block these neuroendocrine transcription factors.
Synthetic Lethality Approaches to Target Loss of Function Mutations in SCLC Tumor Suppressor Genes: Synthetic lethality provides a paradigm for targeting cancers that have LOF mutations in tumor suppressor genes. In applying this paradigm, one looks for specific vulnerabilities that are created upon loss of the gene of interest. We previously utilized a CRISPR/Cas9 based screening approach in SCLC and discovered that RB1 loss is synthetic lethal with Aurora kinases (A or B), which helped inspire new clinical trials treating SCLC patients with Aurora kinase inhibitors. We are now interested in identifying other synthetic lethal interactors with RB1 and/or TP53 in SCLC and whether the approaches we developed to identify synthetic lethal interactors with RB1 can be applied to other SCLC tumor suppressor genes. Ultimately, we hope to identify synthetic lethal targets with LOF tumor suppressors in SCLC.
CRISPR/Cas9 Genetically-Engineered Mouse Models of SCLC to Study Candidate Target Genes: Based on a previous established genetically-engineered mouse model (GEMM) that is generated by conditionally deleting Rb1, Trp53, and Rbl2 in the lung using Cre-Lox recombination (referred to hereafter as “Traditional RPP GEMM”), we developed a new SCLC GEMM entirely using CRISPR/Cas9. This was done by intratracheally injecting an adenovirus that encodes sgRNAs targeting Rb1, Tp53, and Rbl2 (RPP) and Cre-recombinase into lox-stop-lox-Cas9 (LSL-Cas9) mice (referred to hereafter as “CRISPR RPP GEMM”) to induce in vivo CRISPR/Cas9 editing of somatic cells in the lung. A major advantage of our CRISPR RPP GEMM is that our CRISPR RPP GEMM allows for genetic inactivation of candidate target genes (“T”) using CRISPR by introducing “T” sgRNAs along with the RPP sgRNAs. We initially used this approach to study the consequences of inactivating the candidate dependency Kdm5a during SCLC tumorigenesis and demonstrated that Kdm5a functions to promote SCLC tumorigenesis and metastasis. We have now applied this approach to study other known mutated SCLC tumor suppressors including NOTCH1 and NOTCH2 and the SCLC oncogenic neuroendocrine transcription factor ASCL1.
Our laboratory utilizes this CRISPR-based GEMM to study how inactivation of new candidate target genes (either novel candidate oncoproteins identified using the CRISPR/Cas9 screening approaches described above or tumor suppressor genes that are mutated in human SCLC) impact SCLC tumorigenesis. We have and are currently using this immunocompetent GEMM (and a syngeneic model derived from these SCLC GEMM tumors) to study how novel therapeutic approaches impact tumor immunogenicity and the tumor immune microenvironment.