Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing.

Transacting siRNA (tasiRNA) biogenesis in Arabidopsis is initiated by microRNA (miRNA) -guided cleavage of primary transcripts. In the case of TAS3 tasiRNA formation, ARGONAUTE7 (AGO7)-miR390 complexes interact with primary transcripts at two sites, resulting in recruitment of RNA-DEPENDENT RNA POLYMERASE6 for dsRNA biosynthesis. An extensive screen for Arabidopsis mutants with specific defects in TAS3 tasiRNA biogenesis or function was done. This yielded numerous ago7 mutants, one dcl4 mutant, and two mutants that accumulated low levels of miR390. A direct genome sequencing-based approach to both map and rapidly identify one of the latter mutant alleles was developed. This revealed a G-to-A point mutation (mir390a-1) that was calculated to stabilize a relatively nonpaired region near the base of the MIR390a foldback, resulting in misprocessing of the miR390/miR390* duplex and subsequent reduced TAS3 tasiRNA levels. Directed substitutions, as well as analysis of variation at paralogous miR390-generating loci (MIR390a and MIR390b), indicated that base pair properties and nucleotide identity within a region 4-6 bases below the miR390/miR390* duplex region contributed to the efficiency and accuracy of precursor processing.

Loss of p53 expression in cancer cells alters cell cycle response after inhibition of exportin-1 but does not prevent cell death.

The tumor suppressor protein p53 is central to the cellular stress response and may be a predictive biomarker for cancer treatments. Upon stress, wildtype p53 accumulates in the nucleus where it enforces cellular responses, including cell cycle arrest and cell death. p53 is so dominant in its effects, that p53 enforcement - or - restoration therapy is being studied for anti-cancer therapy. Two mechanistically distinct small molecules that act via p53 are the selective inhibitor of nuclear export, selinexor, and MDM2 inhibitor, nutlin-3a. Here, individual cells are studied to define cell cycle response signatures, which captures the variability of responses and includes the impact of loss of p53 expression on cell fates. The individual responses are then used to build the population level response. Matched cell lines with and without p53 expression indicate that while loss-of-function results in altered cell cycle signatures to selinexor treatment, it does not diminish overall cell loss. On the contrary, response to single-agent nutlin-3a shows a strong p53-dependence. Upon treatment with both selinexor and nutlin-3a there are combination effects in at least some cell lines - even when p53 is absent. Collectively, the findings indicate that p53 does act downstream of selinexor and nutlin-3a, and that p53 expression is dispensable for selinexor to cause cell death, but nutlin-3a response is more p53-dependent. Thus, TP53 disruption and lack of expression may not predict poor cell response to selinexor, and selinexor's mechanism of action potentially provides for strong efficacy regardless of p53 function.

Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers.

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor.

Longitudinal tracking is a powerful approach to understand the biology of single cells. In cancer therapy, outcome is determined at the molecular and cellular scale, yet relationships between cellular response and cell fate are often unknown. The selective inhibitor of nuclear export, selinexor, is in development for the treatment of various cancers. Selinexor covalently binds exportin-1, causing nuclear sequestration of cargo proteins, including key regulators of the cell cycle and apoptosis. The cell cycle effects of selinexor and the relationships between cell cycle effects and cell fates, has not been described for individual cells. Using fluorescent cell cycle indicators we report the majority of cell death after selinexor treatment occurs from a protracted G1-phase and early S-phase. G1- or early S-phase treated cells show the strongest response and either die or arrest, while those treated in late S- or G2-phase progress to mitosis and divide. Importantly, the progeny of cell divisions also die or arrest, mostly in the next G1-phase. Cells that survive selinexor are negative for multiple proliferation biomarkers, indicating a penetrant, arrested state. Selinexor acts quickly, shows strong cell cycle selectivity, and is highly effective at arresting cell growth and inducing death in cancer-derived cells.

A potential therapeutic strategy for chronic lymphocytic leukemia by combining Idelalisib and GS-9973, a novel spleen tyrosine kinase (Syk) inhibitor.

Agents that target B-cell receptor (BCR) signaling in lymphoid malignancies including idelalisib (GS-1101) and fostamatinib which inhibit the delta isoform of PI3 kinase (PI3Kd) and spleen tyrosine kinase (Syk) respectively have shown significant clinical activity. By disrupting B-cell signaling pathways, idelalisib treatment has been associated with a dramatic lymph node response, but eradication of disease and relapse in high risk disease remain challenges. Targeting the BCR signaling pathway with simultaneous inhibition of PI3Kd and Syk has not yet been reported. We evaluated the pre-clinical activity of idelalisib combined with the novel and selective Syk inhibitor GS-9973 in primary peripheral blood and bone marrow Chronic Lymphocytic Leukemia (CLL) samples. Both PI3Kd and Syk inhibition reduced CLL survival and in combination induced synergistic growth inhibition and further disrupted chemokine signaling at nanomolar concentrations including in bone marrow derived and poor risk samples. Simultaneous targeting of these kinases may significantly increase clinical activity.

Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis.

RNA interference pathways can involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNAs (miRNAs). Here, Arabidopsis thaliana secondary siRNAs from mRNA as well as trans-acting siRNAs are shown to be triggered through initial targeting by a 22-nucleotide (nt) miRNA that associates with AGO1. In contrast to canonical 21-nt miRNAs, 22-nt miRNAs primarily arise from foldback precursors containing asymmetric bulges. Using artificial miRNA constructs, conversion of asymmetric foldbacks to symmetric foldbacks resulted in the production of 21-nt forms of miR173, miR472 and miR828. Both 21- and 22-nt forms associated with AGO1 and guided accurate slicer activity, but only 22-nt forms were competent to trigger RDR6-dependent siRNA production from target RNA. These data suggest that AGO1 functions differentially with 21- and 22-nt miRNAs to engage the RDR6-associated amplification apparatus.