Lung cancer is a highly metastatic disease. Although commonly considered to be a late event in disease pathogenesis, micrometastasis may also occur as an early phenomenon. Induction of epithelial-mesenchymal transition (EMT) is associated with changes in mechanical properties, predominantly due to increased cell contractility and actin stress fiber formation. The molecular mechanisms regulating actin dynamics during EMT in premalignant cells have not yet been defined. Using a novel constricted migration selection strategy and physomic techniques, including deformability cytometry and atomic force microscopy, we identified a highly motile (HM) subpopulation of HBECs, with enhanced heritable migratory capacity both in vitro and in vivo. The HBECs used for selection were modified with genetic changes that can be found in premalignancy (p53null, activated Kras-G12D). Thus, this sub-population of HM-HBECs offer a unique model to investigate premalignant cell migration. Comparative RNA-seq datasets and confocal live cell imaging technology reveal increased migration and expression of key EMT genes in HM-HBECs. HM-HBECs cells were found to be characterized by the transient accumulation of actin stress fibers as compared to parental-HBECs. We performed high-throughput kinase inhibitor screening to better understand the role of kinases regulating early HBEC migration. Incucyte based secondary screen assays established that ERK-MEK pathway inhibition plays a key role in inhibiting actin stress fiber formation and actin-associated protein network assembly thereby delaying early migration. In the absence of a sensitive quantitative method to detect actin cytoskeleton remodeling in a non-destructive manner, we developed computational methods to extract and quantify the actin stress fibers from Super Resolution Optical Fluctuation imaging (SOFI) and confocal microscopic images. SOFI-based fluorescence imaging based on temporal, stochastic “on” and “off” fluorescence fluctuations in combination with actin-tagged blinking fluorescent proteins such as Dronpa showed advantages over methods that use fixed samples in studying actin filament assembly and disassembly. We extracted the orientations of the fibers and the width of their distribution in each time point to quantitatively distinguish different architectures. Our preliminary data suggest a Rac1-Cortactin-actin dynamic regulatory axis that may lead to enhanced migration. This is accompanied by the orientation of actin fibers favoring EMT. Future studies are anticipated to identify novel targets in premalignancy that could be exploited for lung cancer interception. M.K.P. and A.B. contributed equally to this workCitation Format: Manash K. Paul, Arkaprabha Basu, Bharti Bisht, Paul Pagano, Yari Fontebasso, Kostyantyn Krysan, Linh Tran, Mitchel Alioscha-Perez, John Minna, Dino DiCarlo, Hichem Sahli, Shimon Weiss, Steven M. Dubinett. Regulation and role of actin dynamics in promoting premalignant cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1030.