Mounting evidence supports a role for the immune system in breast cancer outcomes. The ability to distinguish highly immunogenic tumors susceptible to anti-tumor immunity from weakly immunogenic or inherently immune-resistant tumors would guide development of therapeutic strategies in breast cancer. Genomic, transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were used to examine statistical associations between tumor mutational burden (TMB) and the survival of patients whose tumors were assigned to previously-described prognostic immune subclasses reflecting favorable, weak or poor immune-infiltrate dispositions (FID, WID or PID, respectively). Tumor immune subclasses were associated with survival in patients with high TMB (TMB-Hi, P < 0.001) but not in those with low TMB (TMB-Lo, P = 0.44). This statistical relationship was confirmed in the METABRIC cohort (TMB-Hi, P = 0.047; TMB-Lo, P = 0.39), and also found to hold true in the more-indolent Luminal A tumor subtype (TMB-Hi, P = 0.011; TMB-Lo, P = 0.91). In TMB-Hi tumors, the FID subclass was associated with prolonged survival independent of tumor stage, molecular subtype, age and treatment. Copy number analysis revealed the reproducible, preferential amplification of chromosome 1q immune-regulatory genes in the PID immune subclass. These findings demonstrate a previously unappreciated role for TMB as a determinant of immune-mediated survival of breast cancer patients and identify candidate immune-regulatory mechanisms associated with immunologically cold tumors. Immune subtyping of breast cancers may offer opportunities for therapeutic stratification.

Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentation. Using a sensitized mutagenesis screen, we identified Smarca4 as a modifier gene that exacerbates the phenotypic severity of Sox10 haplo-insufficient mice. Conditional deletion of Smarca4 in SOX10 expressing cells resulted in reduced numbers of cranial and ventral trunk melanoblasts. To define the requirement for the Smarca4 -encoded BRG1 subunit of the SWI/SNF chromatin remodeling complex, we employed in vitro models of melanocyte differentiation in which induction of melanocyte-specific gene expression is closely linked to chromatin alterations. We found that BRG1 was required for expression of Dct, Tyrp1 and Tyr, genes that are regulated by SOX10 and MITF and for chromatin remodeling at distal and proximal regulatory sites. SOX10 was found to physically interact with BRG1 in differentiating melanocytes and binding of SOX10 to the Tyrp1 distal enhancer temporally coincided with recruitment of BRG1. Our data show that SOX10 cooperates with MITF to facilitate BRG1 binding to distal enhancers of melanocyte-specific genes. Thus, BRG1 is a SOX10 co-activator, required to establish the melanocyte lineage and promote expression of genes important for melanocyte function.

SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to directly activate genes that encode components of peripheral myelin.