Understanding how cell fate decisions are regulated is a fundamental goal of developmental and stem cell biology. Most studies on the control of cell fate decisions address the contributions of changes in transcriptional programming, epigenetic modifications, and biochemical differentiation cues. However, recent studies have found that other aspects of cell biology also make important contributions to regulating cell fate decisions. These cues can have a permissive or instructive role and are integrated into the larger network of signaling, functioning both upstream and downstream of developmental signaling pathways. Here, we summarize recent insights into how cell fate decisions are influenced by four aspects of cell biology: metabolism, reactive oxygen species (ROS), intracellular pH (pHi), and cell morphology. For each topic, we discuss how these cell biological cues interact with each other and with protein-based mechanisms for changing gene transcription. In addition, we highlight several questions that remain unanswered in these exciting and relatively new areas of the field.

Many lysosome functions are determined by a lumenal pH of ∼5.0, including the activity of resident acid-activated hydrolases. Lysosome pH (pHlys) is often increased in neurodegenerative disorders and predicted to be decreased in cancers, making it a potential target for therapeutics to limit the progression of these diseases. Accurately measuring pHlys, however, is limited by currently used dyes that accumulate in multiple intracellular compartments and cannot be propagated in clonal cells for longitudinal studies or used for in vivo determinations. To resolve this limitation, we developed a genetically encoded ratiometric pHlys biosensor, pHLARE (pHLysosomal Activity REporter), which localizes predominantly in lysosomes, has a dynamic range of pH 4.0 to 6.5, and can be stably expressed in cells. Using pHLARE we show decreased pHlys with inhibiting activity of the mammalian target of rapamycin complex 1 (mTORC1), in breast, pancreatic, colon, and glioblastoma cancer cells compared with untransformed cells, and with the activated oncogenes H-RasV12 and R-RasV12. pHLARE is a new tool to accurately measure pHlys, for improved understanding of lysosome dynamics that could be a promising therapeutic target.

The role of distinct actin filament architectures in epithelial plasticity remains incompletely understood. We therefore determined roles for formins and the Arp2/3 complex, which are actin nucleators generating unbranched and branched actin filaments, respectively, in the process of epithelial to mesenchymal transition (EMT). In clonal lung, mammary, and renal epithelial cells, the formin activity inhibitor SMIFH2 but not the Arp2/3 complex activity inhibitor CK666 blocked EMT induced by TGF-β. SMIFH2 prevented the proximal signal of increased Smad2 phosphorylation and hence also blocked downstream EMT markers, including actin filament remodeling, decreased expression of the adherens junction protein E-cadherin, and increased expression of the matrix protein fibronectin and the transcription factor Snail. The short hairpin RNA silencing of formins DIAPH1 and DIAPH3 but not other formins phenocopied SMIFH2 effects and inhibited Smad2 phosphorylation and changes in Snail and cadherin expression. Formin activity was not necessary for the cell surface expression or dimerization of TGF-β receptors, or for nuclear translocation of TAZ, a transcription cofactor in Hippo signaling also regulated by TGF-β. Our findings reveal a previously unrecognized role for formin-dependent actin architectures in proximal TGF-β signaling that is necessary for Smad2 phosphorylation but not for cross-talk to TAZ.

Olaparib is a potent orally bioavailable poly(ADP-ribose) polymerase inhibitor (PARPi), approved for BRCA-mutated ovarian and breast cancers. We recently showed that olaparib at clinically achievable concentrations exerts anti-proliferative and pro-apoptotic effects in vitro as monotherapy against primary acute myeloid leukemia (AML) blasts, while sparing normal bone marrow (BM) hematopoietic cells. Since AML expresses low levels of death receptors that may contribute to apoptosis resistance, in this study we investigated whether the anti-leukemia activity of olaparib involves modulation of FAS and TRAIL receptors DR5 and DR4. Our data show that the primary AML samples tested express FAS and DR5 transcripts at levels lower than normal BM. In this context, apoptosis triggered by olaparib is associated with a dose-dependent up-regulation of death receptors expression and caspase 8 activation. Olaparib-mediated FAS up-regulation requires NF-κB activation, as indicated by the increase of p65 phosphorylation and decrease of IκBα. Moreover, FAS up-regulation is abrogated by pretreatment of AML cells with two different NF-κB inhibitors. These results indicate that NF-κB activation and consequent induction of death receptor expression contribute to the anti-leukemia effect of olaparib in AML.

Myelodysplastic syndromes (MDS) are highly heterogeneous myeloid diseases, characterized by frequent genetic/chromosomal aberrations. Olaparib is a potent, orally bioavailable poly(ADP-ribose) polymerase 1 (PARP1) inhibitor with acceptable toxicity profile, designed as targeted therapy for DNA repair defective tumors. Here, we investigated olaparib activity in primary cultures of bone marrow mononuclear cells collected from patients with MDS (n = 28). A single treatment with olaparib induced cytotoxic effects in most samples, with median IC50 of 5.4 µM (2.0–24.8 µM), lower than plasma peak concentration reached in vivo. In addition, olaparib induced DNA damage as shown by a high proportion of γH2AX positive cells in samples with low IC50s. Olaparib preferentially killed myeloid cells causing a significant reduction of blasts and promyelocytes, paralleled by an increase in metamyelocytes and mature granulocytes while sparing lymphocytes that are not part of the MDS clone. Consistently, flow cytometry analysis revealed a decrease of CD117+/CD123+ immature progenitors (p < 0.001) and induction of CD11b+/CD16+ (p < 0.001) and CD10+/CD15+ (p < 0.01) neutrophils. Morphological and immunophenotypic changes were associated with a dose-dependent increase of PU.1 and CEBPA transcription factors, which are drivers of granulocytic and monocytic differentiation. Moreover, the combination of olaparib with decitabine resulted in augmented cytotoxic and differentiating effects. Our data suggest that olaparib may have therapeutic potential in MDS patients.