Background: CDK4/6 inhibitors (CDKi) have improved disease control in hormone-receptor-positive, HER2-negative metastatic breast cancer, but most patients develop progressive disease. Methods: We asked whether host stromal senescence after CDK4/6 inhibition affects metastatic seeding and growth of CDKi-resistant mammary cancer cells by using the p16-INK-ATTAC mouse model of inducible senolysis. Results: Palbociclib pretreatment of naïve mice increased lung seeding of CDKi-resistant syngeneic mammary cancer cells, and this effect was reversed by depletion of host senescent cells. RNA sequencing analyses of lungs from non-tumor-bearing p16-INK-ATTAC mice identified that palbociclib downregulates immune-related gene sets and gene expression related to leukocyte migration. Concomitant senolysis reversed a portion of these effects, including pathway-level enrichment of TGF-β- and senescence-related signaling. CIBERSORTx analysis revealed that palbociclib alters intra-lung macrophage/monocyte populations. Notably, lung metastases from palbociclib-pretreated mice revealed senescent endothelial cells. Palbociclib-treated endothelial cells exhibit hallmark senescent features in vitro, upregulate genes involved with the senescence-associated secretory phenotype, leukocyte migration, and TGF-β-mediated paracrine senescence and induce tumor cell migration and monocyte trans-endothelial invasion in co-culture. Conclusions: These studies shed light on how stromal senescence induced by palbociclib affects lung metastasis, and they describe palbociclib-induced gene expression changes in the normal lung and endothelial cell models that correlate with changes in the tumor microenvironment in the lung metastatic niche.

The Hippo pathway signaling mediated through YAP/TAZ, and the transcription factor TEAD is known to be involved in primary tumor progression. Here we report that novel TEAD inhibitors (iTEAD) cause a significant reduction in the outgrowth of lung metastases from triple negative breast cancer (TNBC) models mediated predominantly through changes in stromal immune signaling. TEAD inhibition did not affect the proliferation of TNBC cancer cells in vitro or the growth of the primary tumor in vivo . In normal mice that were treated with iTEAD in the absence of tumors, the lungs showed a decrease in pro-tumor inflammatory pathways. However, the IL12 signaling pathway was enhanced and its production from isolated lung tissue resident macrophages, but not bone marrow derived macrophages, was elevated. In syngeneic TNBC mouse models, inhibition of TEAD suppressed pro-tumor inflammation and the M2-like macrophage phenotype in lung tissues, and increased the infiltration of CD8+ T cells into the lung as well as Th1 CD4+ T cells, restoring an immune responsive microenvironment. iTEAD-treated T cells showed enhanced cytotoxicity and degranulation when co-cultured with cancer cells via increased IL-2 activity. Furthermore, TEAD inhibition or knockdown, enhanced T-cell macrophage crosstalk and anti-tumor activity in 3D tumorspheres which was reversed by IL12 neutralizing antibodies. Our data supports a multifaceted model of TEAD inhibition on the innate and adaptive immune cells as they respond to tumor cell signals and reveals an important stromal phenotype by which TEAD inhibitors could reverse immune suppression and eliminate seeded metastases in the lungs.

Background. Ductal carcinoma in situ (DCIS) is a non-invasive disease of the breast. This study assessed the impact of the CDK4/6 inhibitor palbociclib on halting cell cycle progression in DCIS between diagnostic biopsy and surgical resection of the lesion. Methods. A dual-arm study was conducted involving 17 participants with DCIS who were given either 100 mg of palbociclib PO daily for twelve days or no treatment before surgery. FFPE tissues from the diagnostic biopsy and from surgical specimens were analyzed to assess changes due to treatment. Results. Palbociclib treatment significantly reduced Ki67 staining in surgical tissues. RNA-seq analysis comparing the diagnostic biopsy and surgical specimens showed significant downregulation of cellular proliferation pathways after treatment. A cellular deconvolution analysis demonstrated a non-significant trend of a treatment-induced decrease in the abundance of mature luminal epithelial cells. Conclusions. Short-term palbociclib monotherapy before surgical resection was safe and well tolerated in participants. Tissue analysis showed a significant reduction in proliferation markers and the downregulation of cell cycle progression genes within DCIS. These results support further investigation of palbociclib in patients with DCIS. Trial registration. ClinicalTrials.gov NCT03535506.