Functional Characterization of SMARCA4 Variants Identified by Targeted Exome-Sequencing of 131,668 Cancer Patients

In human primary tumors and tumor-derived cell lines, more than 20% of cancer genomes harbor mutations in one or more BAF subunits, and alterations in the core catalytic subunit SMARCA4 have been identified across multiple tumor types. Because cells lacking SMARCA4 depend on its paralog SMARCA2, SMARCA2 has emerged as an attractive therapeutic target. However, existing genomic studies have characterized SMARCA4 mutations using only limited patient datasets and have failed to assess differences in their occurrence across other BAF subunits and oncogenic driver genes. Therefore, before translating potential SMARCA2-targeted therapies into clinical applications, we must elucidate the mutational spectrum of SMARCA4 and its functional characteristics.

Recently,A Joint Team from Genentech and Foundation Medicine Reports a Genomic Landscape of Solid Tumors Based on 131,668 Cancer Patients. The study identified one or more SMARCA4 gene mutations in 9,434 patients. Among these, homozygous SMARCA4 mutations were highly prevalent in certain tumor types and were associated with reduced patient survival. Furthermore, the study revealed previously undescribed hotspot missense mutations in the SMARCA4 helicase domain, with some SMARCA4 missense mutations partially or completely escaping paralog dependency.The research findings were published inNature Communicationsabove, the article is titled “Functional characterization of SMARCA4 variants identified by targeted exome-sequencing of 131,668 cancer patients”.

The article was published inNature CommunicationsUpper

The research team employed targeted exome sequencing to analyze solid tumors in 131,668 cancer patients, thereby generating a genomic atlas. The analysis revealed SMARCA4 alterations in 9,434 patients, ultimately identifying 10,562 SMARCA4 variants, of which more than half (6,289) were missense mutations. Furthermore,SMARCA4 Mutations Occur in Multiple Cancer Types with a Frequency of Up to 16%(Figure 1), which is consistent with the mutational profiles described in The Cancer Genome Atlas (TCGA) and other pan-cancer analyses. For certain cancers, such as non-small cell lung cancer (NSCLC) and cancer of unknown primary (CUP), the incidence of SMARCA4 homozygous mutations is high, likely due to the high frequency of SMARCA4 loss of heterozygosity (LOH) observed in patients with NSCLC (77%) and CUP (68%). Meanwhile, researchers assessed whether SMARCA4 mutations co-occur with alterations in other actionable driver genes.The results showed that SMARCA4 mutations are mutually exclusive with the most prevalent targetable oncogenes (EGFR, ALK, MET, ROS1, and RET) in NSCLC., and EGFR mutations exhibited the strongest mutual exclusivity with SMARCA4 mutations.

Figure 1. SMARCA4 mutation profile in the FoundationCORE® patient cohort, Source:Nature Communications

To investigate whether SMARCA4 mutations are associated with differences in clinical prognosis, researchers conducted a retrospective study of patients with advanced non-small cell lung cancer (NSCLC) (stage ≥IIIB) who had undergone tumor profiling using FoundationOne® or FoundationOne®CDX. The results showed thatCompared with patients carrying wild-type (WT) SMARCA4, those with non-small cell lung cancer (NSCLC) harboring homozygous truncating SMARCA4 mutations had significantly reduced overall survival. Among SMARCA4-mutant patients who received checkpoint immunotherapy (CIT) during treatment, NSCLC patients with homozygous truncating SMARCA4 mutations had significantly shorter overall survival than WT patients.(Figure 2). The study results indicate that,Unmet Needs in Patients with Advanced NSCLC Harboring Homozygous Truncating SMARCA4 Mutations, Who May Not Benefit from Currently Available Targeted Molecular Therapies and CIT

Figure 2. Homozygous SMARCA4 mutations in NSCLC, Source:Nature Communications

Given that understanding the breadth and functional impact of SMARCA4 mutations is critical for developing therapeutic strategies for related tumors, researchers analyzed 6,289 missense mutations in the SMARCA4 gene. The results revealed thatHotspot SMARCA4 missense mutations occur within the ATP-binding cleft, DNA-binding region, and supporting helix(Figure 3). To better understand the consequences of SMARCA4 missense mutations, researchers functionally characterized a set of mutations identified in the SNF2 C-terminal helicase domain. The results showed that,Only wild-type SMARCA4 was able to remodel nucleosomes in this study, indicating that the mutants significantly reduced detectable nucleosome remodeling activity.

Figure 3. Hotspot missense mutations in the SMARCA4 ATPase helicase domain, Source:Nature Communications

The ability of SMARCA2 to compensate for SMARCA4 loss makes SMARCA2 an attractive therapeutic target in SMARCA4-mutant tumor types, which has significant implications for the future clinical development of SMARCA2-targeted therapies. However, it remains unclear whether SMARCA4 missense mutations can compensate for SMARCA2 loss.This study found that SMARCA4 mutants (A1186T, R973L) could rescue cell growth to varying degrees after SMARCA2 knockout.The research team discovered through ATAC-seq analysis that SMARCA2 depletion led to a significant decrease in chromatin accessibility. Wild-type SMARCA4 (SMARCA4 WT) fully restored chromatin accessibility, while the A1186T and R973L mutants demonstrated the ability to overcome the loss of accessibility. Furthermore, the endogenous SMARCA4 mutant (A1186T) retained sufficient activity in the absence of SMARCA2. These experimental results indicate thatSMARCA4 Missense Mutations Maintain SMARCA2 Deficiency to Varying Degrees

In summary, this study employed techniques such as ATAC-seq, ChIP-seq, and RNA-seq to analyze solid tumors from clinical cancer patients, thereby characterizing the functional features of SMARCA4 mutants. In light of the ongoing development of SMARCA2 inhibitors, this comprehensive exploration of SMARCA4 mutants provides important insights for formulating and selecting therapeutic strategies for future cancer patients.

References:

Functional characterization of SMARCA4 variants identified by targeted exome-sequencing of 131,668 cancer patients. Nature Communications, 2020  https://doi.org/10.1038/s41467-020-19402-8