THERAPEUTIC VULNERABILITIES
Using the integrative mouse model, we demonstrated that select genomic subtypes of gastric and esophageal cancer are susceptible to DNA damage response (DDR) inhibitors. Based on these results, we are pursing preclinical testing of DDR pathway inhibitors to investigate the therapeutic efficacy of these agents alone and in combination with specific chemotherapeutic agents.
Background: Disrupting the balance between stem cell and differentiation programs is a defining property of colorectal cancer (CRC). Genomic alterations that block intestinal differentiation - either through activation of stem cell-like programs or suppression of differentiation pathways – play a central role in CRC pathogenesis. Our group previously identified that SOX9 is a key mediator of impaired differentiation by mediating aberrant stem cell-like activity in CRC (Liang et al., Gastroenterology 2022). Keratin 20 (KRT20) is a well-established marker of differentiated intestinal cells, notably absent in normal stem cells, and suppressed in models of cancer initiation. Applying CRISPR screens to our system, we nominated epigenetic factors that regulate stem cell and differentiation programs in CRC.
Findings: To systematically identify functional regulators of aberrant stem cell-like and differentiation states in CRC, we engineered single and dual endogenous reporter systems by knocking-in fluorescent probes at SOX9 and KRT20 genomic loci. This innovative platform enabled dynamic tracking of stem cell-like and differentiation activity in real time. To optimize the system, we conducted focused genetic screens to assess key variables, including perturbation strategies (CRISPR versus shRNA), screen duration, cell line suitability, reporter configuration (single versus dual), and fluorescent probes (swapping markers to ensure reproducibility). We then applied a CRISPR-based discovery screen targeting 78 epigenetic regulators with 542 sgRNAs to this platform, identifying factors whose loss of function altered stem cell-like activity and differentiation programs. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominated SMARCB1 of the chromatin remodeling BAF complex (also known as SWI/SNF) as a negative regulator of CRC differentiation. SMARCB1 suppression activated differentiation in a patient-derived CRC organoid model by bulk RNA-sequencing. Further functional studies established SMARCB1 as a key dependency across CRC cell lines, with its loss significantly impairing tumor growth in multiple human CRC models in vivo.
Significance: Our study underscores the power of applying genetic screens to biologically relevant functional platforms for uncovering regulators of disease-specific phenotypes. By utilizing endogenous reporter systems, we identified key epigenetic factors driving aberrant stem cell-like and differentiation programs in CRC. Notably, our identification of SMARCB1 as a central repressor of intestinal differentiation provides new insights into the epigenetic control of CRC biology. These findings open new avenues for exploring SMARCB1 and related chromatin remodeling factors as therapeutic targets in colorectal cancer.
Spisak*, Chen*, Likasitwatanakul*, Doan* et al., Nature Communications 2024
Findings: To systematically identify functional regulators of aberrant stem cell-like and differentiation states in CRC, we engineered single and dual endogenous reporter systems by knocking-in fluorescent probes at SOX9 and KRT20 genomic loci. This innovative platform enabled dynamic tracking of stem cell-like and differentiation activity in real time. To optimize the system, we conducted focused genetic screens to assess key variables, including perturbation strategies (CRISPR versus shRNA), screen duration, cell line suitability, reporter configuration (single versus dual), and fluorescent probes (swapping markers to ensure reproducibility). We then applied a CRISPR-based discovery screen targeting 78 epigenetic regulators with 542 sgRNAs to this platform, identifying factors whose loss of function altered stem cell-like activity and differentiation programs. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominated SMARCB1 of the chromatin remodeling BAF complex (also known as SWI/SNF) as a negative regulator of CRC differentiation. SMARCB1 suppression activated differentiation in a patient-derived CRC organoid model by bulk RNA-sequencing. Further functional studies established SMARCB1 as a key dependency across CRC cell lines, with its loss significantly impairing tumor growth in multiple human CRC models in vivo.
Significance: Our study underscores the power of applying genetic screens to biologically relevant functional platforms for uncovering regulators of disease-specific phenotypes. By utilizing endogenous reporter systems, we identified key epigenetic factors driving aberrant stem cell-like and differentiation programs in CRC. Notably, our identification of SMARCB1 as a central repressor of intestinal differentiation provides new insights into the epigenetic control of CRC biology. These findings open new avenues for exploring SMARCB1 and related chromatin remodeling factors as therapeutic targets in colorectal cancer.
Spisak*, Chen*, Likasitwatanakul*, Doan* et al., Nature Communications 2024