Cells continuously sense and respond to external mechanical forces through their cytoskeleton. Here we show that only a small subset of actin fibers, those forming the perinuclear actin cap that wraps around the nucleus, form in response to low physiological mechanical stresses in adherent fibroblasts. While conventional basal stress fibers form only past a threshold shear stress of 0.5 dyn/cm2, actin-cap fibers are formed at shear stresses 50 times lower and orders-of-magnitude faster than biochemical stimulation. This fast differential response is uniquely mediated by focal adhesion protein zyxin at low shear stress and actomyosin fibers of the actin cap. We identify additional roles for lamin A/C of the nuclear lamina and linkers of nucleus to cytoskeleton (LINC) molecules nesprin2giant and nesprin3, which anchor actin cap fibers to the nucleus. These results suggest an interconnected physical pathway for mechanotransduction, from the extracellular milieu to the nucleus.

The perinuclear actin cap (or actin cap) is a recently characterized cytoskeletal organelle composed of thick, parallel, and highly contractile acto-myosin filaments that are specifically anchored to the apical surface of the interphase nucleus. The actin cap is present in a wide range of adherent eukaryotic cells, but is disrupted in several human diseases, including laminopathies and cancer. Through its large terminating focal adhesions and anchorage to the nuclear lamina and nuclear envelope through LINC complexes, the perinuclear actin cap plays a critical role both in mechanosensation and mechanotransduction, the ability of cells to sense changes in matrix compliance and to respond to mechanical forces, respectively.

MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, believed to be the driving factor in the pathogenesis of this MM subtype. MMSET overexpression in MM leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3), as well as changes in proliferation, gene expression, and chromatin accessibility. Prior work linked methylation of histones to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET may play a role in DNA damage repair and response. In U2OS cells, we found that MMSET is required for efficient non-homologous end joining as well as homologous recombination. Loss of MMSET led to loss of expression of several DNA repair proteins, as well as decreased recruitment of DNA repair proteins to sites of DNA double strand breaks (DSBs). Using genetically matched MM cell lines that had either high (pathological) or low (physiological) expression of MMSET, we found that MMSET high cells had increased damage at baseline. Upon addition of a DNA damaging agent, MMSET high cells repaired DNA damage at an enhanced rate and continued to proliferate, whereas MMSET low cells accumulated DNA damage and entered cell cycle arrest. In a murine xenograft model using t(4;14)+ KMS11 MM cells harboring an inducible MMSET shRNA, depletion of MMSET enhanced the efficacy of chemotherapy, inhibiting tumor growth and extending survival. These findings help explain the poorer prognosis of t(4;14) MM and further validate MMSET as a potential therapeutic target in MM and other cancers.

Heterogeneity of cellular phenotypes in asynchronous cell populations placed in the same biochemical and biophysical environment may depend on cell cycle and chromatin modifications; however, no current method can measure these properties at single-cell resolution simultaneously and in situ. Here, we develop, test, and validate a new microscopy assay that rapidly quantifies global acetylation on histone H3 and measures a wide range of cell and nuclear properties, including cell and nuclear morphology descriptors, cell-cycle phase, and F-actin content of thousands of cells simultaneously, without cell detachment from their substrate, at single-cell resolution. These measurements show that isogenic, isotypic cells of identical DNA content and the same cell-cycle phase can still display large variations in H3 acetylation and that these variations predict specific phenotypic variations, in particular, nuclear size and actin cytoskeleton content, but not cell shape. The dependence of cell and nuclear properties on cell-cycle phase is assessed without artifact-prone cell synchronization. To further demonstrate its versatility, this assay is used to quantify the complex interplay among cell cycle, epigenetic modifications, and phenotypic variations following pharmacological treatments affecting DNA integrity, cell cycle, and inhibiting chromatin-modifying enzymes.

Disease progression and drug response may vary significantly from patient to patient. Fortunately, the rapid development of high-throughput ‘omics’ technologies has allowed for the identification of potential biomarkers that may aid in the understanding of the heterogeneities in disease development and treatment outcomes. However, mechanistic gaps remain when the genome or the proteome are investigated independently in response to drug treatment. In this article, we discuss the current status of pharmacogenomics in precision medicine and highlight the needs for concordant analysis at the proteome and metabolome levels via the more recently-evolved fields of pharmacoproteomics, toxicoproteomics, and pharmacometabolomics. Integrated ‘omics’ investigations will be critical in piecing together targetable mechanisms of action for both drug development and monitoring of therapy in order to fully apply precision medicine to the clinic.