Unraveling Epigenetic Mechanisms that Regulate Breast Cancer Lung Metastatic Relapse by Rewiring the Immune Microenvironment

Advisor: Jason Huse, MD, PhD

Metastasis accounts for >90% of cancer-related deaths and thwarting metastasis is widely considered to be the 'Holy Grail' of cancer treatment. Breast cancer is the most common cause of cancer-related death among women and is characterized by its proclivity to spread to distant secondary organs like the lungs, and the brain. These disseminated tumor cells lie dormant at the secondary site and can undergo metastatic relapse. Epigenetic alterations are a predominant hallmark of cancer, however, its role in facilitating reawakening of dormant cancer cells and the subsequent metastatic relapse remains to be elucidated. To address this gap of knowledge, we performed an extensive in vivo epigenetic screen in our breast cancer dormancy model to decipher regulators of breast cancer lung metastatic outgrowth. Several subunits of the PBAF complex, a multi-component epigenetic regulator within the SWI/SNF family, emerged as potent suppressors of breast cancer lung metastasis. Amongst them, Brd7 (bromodomain-7), which encodes a PBAF-specific subunit, emerged as the top-scoring hit.

RNAseq revealed that loss-of-Brd7 led to upregulation of oncogenic signaling pathways, like PI3K-AKT-mTOR signaling, EMT transition and Hedgehog signaling. Further, loss-of-Brd7 led to downregulation of antigen presentation, interferon response and MHC class 1 related genes, which suggested that Brd7 loss hindered effective immunosurveillance. ATAC-seq revealed wide-spread shifts in chromatin accessibility upon Brd7-loss, while ChIP-seq demonstrated alteration of H3K27ac labeling at thousands of genomic sites, with Brd7-loss exhibiting reduced H3K27ac binding at multiple transcription factor sites with key roles in immune cell lineages.

Luminex cytokine profiling also revealed to us that loss-of-Brd7 led to upregulation of pro-metastatic cytokine secretion like CXCL10, CCL5, CCL2, CCL20. To further delineate the effect of loss-of-Brd7 on the immune microenvironmental niche, flow cytometric analysis was performed which revealed that loss-of-Brd7 enabled metastasis through the recruitment of pro-tumorigenic neutrophils while downregulating macrophages and dendritic cells in vivo. scRNAseq informed us that while the loss-of-Brd7 led to upregulation of neutrophils, stress response CD4+ T cells and exhausted CD8+ T cells in vivo. Furthermore, therapeutically targeting the loss-of-Brd7 induced breast cancer lung metastasis with an anti-Ly6G neutrophil depleting antibody, Sivelestat (Neutrophil Extracellular Traps inhibitor) and anti-LAG3+anti-CTLA4 immunotherapy regime abrogated the Brd7-KO induced lung metastasis.

Taken together, our findings that BRD7 is a suppressor of breast cancer lung metastasis and a predictive cancer biomarker for lung metastatic relapse , could have major implications in the formulation of paradigm-shifting therapeutic strategies for metastatic breast cancer. We elucidate the intricate mechanisms by which epigenetic regulators-like the PBAF chromatin remodeler subunit BRD7- regulates breast cancer lung metastasis by reprogramming the lung immune microenvironment.

Advisory Committee:
Jason Huse, MD, PhD, Chair
James Allison, PhD
Richard Behringer, PhD
George Calin, MD, PhD
Matthew Gubin, PhD