The generation of force by actomyosin contraction is critical for a variety of cellular and developmental processes. Nonmuscle myosin II is the motor that drives actomyosin contraction, and its activity is largely regulated by phosphorylation of the myosin regulatory light chain. During the formation of the Drosophila cellular blastoderm, actomyosin contraction drives constriction of microfilament rings, modified cytokinesis rings. Here, we find that Drak is necessary for most of the phosphorylation of the myosin regulatory light chain during cellularization. We show that Drak is required for organization of myosin II within the microfilament rings. Proper actomyosin contraction of the microfilament rings during cellularization also requires Drak activity. Constitutive activation of myosin regulatory light chain bypasses the requirement for Drak, suggesting that actomyosin organization and contraction are mediated through Drak’s regulation of myosin activity. Drak is also involved in the maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization. Together, our observations suggest that Drak is the primary regulator of actomyosin dynamics during cellularization.

Summary Src64 is required for actomyosin contraction during cellularization of the Drosophila embryonic blastoderm. The mechanism of actomyosin ring constriction is poorly understood even though a number of cytoskeletal regulators have been implicated in the assembly, organization, and contraction of these microfilament rings. How these cytoskeletal processes are regulated during development is even less well understood. To investigate the role of Src64 as an upstream regulator of actomyosin contraction, we conducted a proteomics screen to identify proteins whose expression levels are controlled by src64. Global levels of actin are reduced in src64 mutant embryos. Furthermore, we show that reduction of the actin isoform Actin 5C causes defects in actomyosin contraction during cellularization similar to those caused by src64 mutation, indicating that a relatively high level of Actin 5C is required for normal actomyosin contraction and furrow canal structure. However, reduction of Actin 5C levels only slows down actomyosin ring constriction rather than preventing it, suggesting that src64 acts not only to modulate actin levels, but also to regulate the actomyosin cytoskeleton by other means.

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KRAS proteins regulate many cellular processes and gain-of-function KRAS mutations constitutively activate effector signal transduction pathways independent of mitogenic stimuli. Oncogenic KRAS-driven cancers also frequently have high glycolytic rates and may have increased sensitivity to metabolic inhibitors. In this study, we characterize a small molecule found to be active in a drug screen that uses transgenic Drosophila melanogaster expressing human KRASG12V in the wing to assess kRAS suppressing activity. The compound partially restored normal wing development in the KRASG12V flies and inhibited growth and signal transduction in multiple oncogenic KRAS-driven cell lines. It also inhibited Complex 1 of the electron transport chain (ETC). Increased glycolysis or addition of exogenous aspartate was sufficient to rescue KRAS function and cellular growth after ETC inhibition. These data indicate that oncogenic KRAS signal transduction requires high cellular ATP and amino acid biosynthesis and suggest a synthetic lethal interaction between KRAS and metabolic targets. We also describe co-mutations that sensitize KRAS-driven cell lines to OxPhos Inhibition and show how differential OxPhos inhibitor sensitivity in lung and pancreatic cancer cell lines can correlate with low and high glycolysis ssGSEA gene signatures. Citation Format: Kanika Sharma, Ming Yi, Giovanna Grandinetti, Ashish B. Chougule, Philip Liaw, Stephen Yanofsky, Solomon Ungashe, Jeffery H. Thomas, William Garland, Matthew Holderfield. ETC inhibitors alter oncogenic KRAS signal transduction [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B08.