Drosophila anion exchanger 2 is required for proper ovary development and oogenesis.

Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type. We found previously that the pHi of cells in the follicle stem cell (FSC) lineage in the Drosophila ovary increases progressively during differentiation from an average of 6.8 in the FSCs, to 7.0 in newly produced daughter cells, to 7.3 in more differentiated cells. Two major regulators of pHi in this lineage are Drosophila sodium-proton exchanger 2 (dNhe2) and a previously uncharacterized gene, CG8177, that is homologous to mammalian anion exchanger 2 (AE2). Based on this homology, we named the gene anion exchanger 2 (ae2). Here, we generated null alleles of ae2 and found that homozygous mutant flies are viable but have severe defects in ovary development and adult oogenesis. Specifically, we find that ae2 null flies have smaller ovaries, reduced fertility, and impaired follicle formation. In addition, we find that the follicle formation defect can be suppressed by a decrease in dNhe2 copy number and enhanced by the overexpression of dNhe2, suggesting that this phenotype is due to the dysregulation of pHi. These findings support the emerging idea that pHi dynamics regulate cell fate decisions and our studies provide new genetic tools to investigate the mechanisms by which this occurs.

Long-term breast cancer response to CDK4/6 inhibition defined by TP53-mediated geroconversion.

Inhibition of CDK4/6 kinases has led to improved outcomes in breast cancer. Nevertheless, only a minority of patients experience long-term disease control. Using a large, clinically annotated cohort of patients with metastatic hormone receptor-positive (HR+) breast cancer, we identify TP53 loss (27.6%) and MDM2 amplification (6.4%) to be associated with lack of long-term disease control. Human breast cancer models reveal that p53 loss does not alter CDK4/6 activity or G1 blockade but instead promotes drug-insensitive p130 phosphorylation by CDK2. The persistence of phospho-p130 prevents DREAM complex assembly, enabling cell-cycle re-entry and tumor progression. Inhibitors of CDK2 can overcome p53 loss, leading to geroconversion and manifestation of senescence phenotypes. Complete inhibition of both CDK4/6 and CDK2 kinases appears to be necessary to facilitate long-term response across genomically diverse HR+ breast cancers.

Therapy-Induced Senescence Contributes to the Efficacy of Abemaciclib in Patients with Dedifferentiated Liposarcoma.

PURPOSE: We conducted research on CDK4/6 inhibitors (CDK4/6i) simultaneously in the preclinical and clinical spaces to gain a deeper understanding of how senescence influences tumor growth in humans. PATIENTS AND METHODS: We coordinated a first-in-kind phase II clinical trial of the CDK4/6i abemaciclib for patients with progressive dedifferentiated liposarcoma (DDLS) with cellular studies interrogating the molecular basis of geroconversion. RESULTS: Thirty patients with progressing DDLS enrolled and were treated with 200 mg of abemaciclib twice daily. The median progression-free survival was 33 weeks at the time of the data lock, with 23 of 30 progression-free at 12 weeks (76.7%, two-sided 95% CI, 57.7%-90.1%). No new safety signals were identified. Concurrent preclinical work in liposarcoma cell lines identified ANGPTL4 as a necessary late regulator of geroconversion, the pathway from reversible cell-cycle exit to a stably arrested inflammation-provoking senescent cell. Using this insight, we were able to identify patients in which abemaciclib induced tumor cell senescence. Senescence correlated with increased leukocyte infiltration, primarily CD4-positive cells, within a month of therapy. However, those individuals with both senescence and increased TILs were also more likely to acquire resistance later in therapy. These suggest that combining senolytics with abemaciclib in a subset of patients may improve the duration of response. CONCLUSIONS: Abemaciclib was well tolerated and showed promising activity in DDLS. The discovery of ANGPTL4 as a late regulator of geroconversion helped to define how CDK4/6i-induced cellular senescence modulates the immune tumor microenvironment and contributes to both positive and negative clinical outcomes. See related commentary by Weiss et al., p. 649.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation.

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+-H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. Our findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms.

Redox-sensitive cross-linking enhances albumin nanoparticle function as delivery system for photodynamic cancer therapy.

Photodynamic cancer therapy is still limited in its efficiency because of a lack of targeted methods avoiding non-specific toxicity. To overcome this we developed a system that is solely effective upon cellular uptake and intracellular activation by incorporating redox-sensitive chemistry. We used a nanoprecipitation method to obtain human serum albumin nanoparticles (HSA NP) with a diameter of 295 ± 5 nm and decorated them with the photosensitizer (PS) chlorin e6 (Ce6). The NP were stabilized using a redox-sensitive cross-linker to create a smart drug delivery system that is activated only upon NP disintegration in the reducing intracellular environment. Indeed, our drug delivery NP broke down in an environment emulating the reducing intracellular environment with 10 mM glutathione, but not under extracellular conditions. In contrast, the control cross-linked with glutaraldehyde did not break down in the reducing environment. Upon NP disintegration Ce6 fluorescence doubled as the result of diminished self-quenching. While the Ce6-HSA NP did not produce a significant amount of singlet oxygen upon irradiation, NP disintegration restored singlet oxygen production to about half of the value generated by the free Ce6. In vitro experiments with HeLa cells showed that the smart system was able to kill up to 81% of the cells while the glutaraldehyde cross-linked control only killed 56% of them at a drug concentration of 10 ng/ml. Also, Ce6 immobilization in HSA NP prevented dark toxicity in three different cell lines. For the first time, we demonstrate that it is possible to design a smart NP drug delivery system delivering a PS drug to cancer cells while avoiding toxicity prior to the uptake and irradiation. This finding may provide a means of designing more efficient PDT in cancer treatment.