Once tumors were established to be growing in the presence of drug (day 36, average tumor size roughly 3 mm 3 mm, n = 16), palbociclib treatment was ceased for 28 days. Levels of miR-432-5p are higher in primary breast cancers demonstrating CDK4/6 resistance compared to those that are sensitive. These data are Furthermore confirmed in pre-treatment and post-progression biopsies from a parotid cancer patient who had responded to ribociclib, demonstrating the clinical relevance of this mechanism. Finally, the CDK4/6 inhibitor resistance phenotype is reversible and by a prolonged drug holiday. Graphical Abstract In Brief Cornell et al. demonstrate a mechanism of acquired CDK4/6 inhibitor resistance that is independent of inherent genetic mutations, is conferred through extracellular signaling, and is reversible and Resistance was mediated by exosomal miRNA, causing increased expression of CDK6 to overcome G1 arrest and promote cell survival. INTRODUCTION Cyclin D-dependent kinase activity is thought to be a driving factor for carcinogenesis in >80% of hormone receptor-positive breast cancers (Massagu, 2004), providing rationale for the inhibition of the cell-cycle kinases, cyclin-dependent kinase 4 (CDK4) and CDK6, in this breast cancer subset (Arnold and Papanikolaou, 2005; Elsheikh et al., 2008; Perou et al., 2000; The Cancer Genome Atlas Network, 2012; Velasco-Velzquez et al., 2011). The use of potent and highly selective CDK4/6 inhibitors, including palbociclib, ribociclib, and abemaciclib, has transformed the treatment of metastatic estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2?) breast cancer based on prolonged progression-free survival when these agents are combined with hormone treatment compared to hormone therapy alone (Cristofanilli et al., 2016; Finn et al., 2016; Goetz et al., 2017; Hortobagyi et al., 2016; FJX1 Sledge et al., 2017). In addition, abemaciclib has been approved as a monotherapy for patients with advanced ER+ breast cancer who have progressed on prior endocrine therapy and chemotherapy (Dickler et al., 2017). CDK4/6 inhibition may also have activity in HER2-driven breast cancer and in triplenegative breast cancers that retain expression of the retinoblastoma (RB) protein (Roberts et al., 2012; Yu et al., 2006). CDK4/6 inhibitor-based treatment is complicated by the development of acquired resistance. To date, resistance mechanisms have not been extensively investigated. In leukemia models, reduced p27Kip1 expression and elevated CDK2 activity can overcome palbociclib-mediated G1 arrest (Wang et al., 2007). In breast cancer models, RB loss, amplification of (Herrera-Abreu et al., 2016), (Yang et al., 2017), or (Formisano et al., 2017) and increased pyruvate dehydrogenase kinase 1 (PDK1) activity (Jansen et al., 2017) are also mechanisms by which the cancer cell can bypass CDK4/6 inhibitor-mediated G1 arrest. In analyses of tumor or liquid biopsies from breast cancer patients treated with CDK4/6 inhibitors, high cyclin E expression may define populations with intrinsic resistance (Turner et al., 2018), while acquired or mutation and fibroblast growth factor receptor (FGFR) pathway activation have been identified in post-progression samples (Condorelli et al., 2018; Formisano et al., 2017; Mao et al., 2018; OLeary et al., 2018). Here, we present a previously unreported mechanism by which resistance to CDK4/6 inhibitor treatment arises. Acquired resistance is centered on increased CDK6 protein concentration as the key determinant, achieved via the suppression of the transforming growth factor (TGF-) pathway mediated by microRNA (miRNA) expression. Consequently, resistance is transmissible by extracellular signaling and is reversible both and and expression in resistant (R100) versus parental cells. These increases in mRNA expression were not accompanied by gene amplification as there was no variation in the copy number of these genes (Figure S2). No significant changes were observed in the remaining Pipendoxifene hydrochloride cyclin and CDK genes (Figure 1C). We also analyzed multiple genes related to cell cycle, growth, and/or CDK4/6 inhibitor resistance (Figure 1D). There Pipendoxifene hydrochloride were significant, albeit small (<2-fold), changes in the expression of and in resistant cells. In correlation with gene expression, the greatest changes in protein expression were increased CDK6 and cyclin D1, observed in both T47D and MCF7 cells, with the expression increasing stepwise in cells that were resistant to higher concentrations of palbociclib (Figure 1E). A small stepwise increase in cyclin E levels was also observed, along with a progressive decrease in CDK1 expression. Phosphorylation of RB at the CDK4/6 site Ser807/Ser811, as well as at Thr356, was maintained in all resistant cells (Figure 1E). CDK6 Knockdown Re-sensitizes Resistant Cells, and Overexpression of CDK6 Confers Resistance in Parental Cells To determine the contribution of CDK6 to palbociclib resistance, we manipulated CDK6 expression in both parental and resistant T47D cells. Neither overexpression of CDK4 or CDK6 nor depletion of CDK6 significantly influenced the cell-cycle profile of parental T47D cells (Figure 2A). Substantial overexpression of CDK4 (CDK4) and CDK6 (CDK6) Pipendoxifene hydrochloride was achieved in parental cells and confirmed by western blot (Figure 2B). In addition, robust knockdown of CDK6 was confirmed in resistant cell lines (Figure 2C). Of.