Managing relapsed refractory lymphoma with palliative oral chemotherapy: A multicentre retrospective study.

PEP-C (prednisolone, etoposide, procarbazine and cyclophosphamide) is an orally administered daily chemotherapy regimen used with palliative intent in relapsed refractory lymphoma. To our knowledge, no data on PEP-C have been reported since the original group described the regimen. Here we present a multicentre retrospective cohort reporting our use of PEP-C in 92 patients over an 8-year period. We find that even heavily pretreated lymphoma can respond to PEP-C, particularly low-grade lymphoma (including mantle cell) and lymphoma that was sensitive to the previous line of systemic therapy (chemosensitive). These characteristics may help in the selection of patients likely to derive benefit. The median overall survival of patients with chemosensitive lymphoma treated with PEP-C is 217 days. Within the limitations of a retrospective cohort, we find that PEP-C is well tolerated: the most common toxicity leading to discontinuation is marrow suppression. We suggest that PEP-C should be considered for patients with relapsed refractory lymphoma in two settings: first, where there is no licensed alternative; and second, where the licensed alternative is an intravenous drug and the patient would prefer to choose an oral chemotherapy option.

In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP3, its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal.

Nuclear localizations of phosphatidylinositol 5-phosphate 4-kinases α and ß are dynamic and independently regulated during starvation-induced stress.

The chicken B-cell line DT40 has two isoforms of phosphatidylinositol 5-phosphate 4-kinase (PI5P4K), α and ß, which are likely to exist as a mixture of obligate homo- and hetero-dimers. Previous work has led us to speculate that an important role of the ß isoform may be to target the more active PI5P4Kα isoform to the nucleus. In the present study we expand upon that work by genomically tagging the PI5P4Ks with fluorochromes in the presence or absence of stable or acute depletions of PI5P4Kß. Consistent with our original hypothesis we find that PI5P4Kα is predominantly (possible entirely) cytoplasmic when PI5P4Kß is stably deleted from cells. In contrast, when PI5P4Kß is inducibly removed within 1 h PI5P4Kα retains its wild-type distribution of approximately 50:50 between cytoplasm and nucleus even through a number of cell divisions. This leads us to speculate that PI5P4Kα is chromatin-associated. We also find that when cells are in the exponential phase of growth PI5P4Kß is primarily cytoplasmic but translocates to the nucleus upon growth into the stationary phase or upon serum starvation. Once again this is not accompanied by a change in PI5P4Kα localization and we show, using an in vitro model, that this is possible because the dimerization between the two isoforms is dynamic. Given this shift in PI5P4Kß upon nutrient deprivation we explore the phenotype of PI5P4K B-null cells exposed to this stress and find that they can sustain a greater degree of nutrient deprivation than their wild-type counterparts possibly as a result of up-regulation of autophagy.

There is no 'Conundrum' of InsP6.

Indirect assays have claimed to quantify phytate (InsP6) levels in human biofluids, but these have been based on the initial assumption that InsP6 is there, an assumption that our more direct assays disprove. We have shown that InsP6 does not and cannot (because of the presence of an active InsP6 phosphatase in serum) exist in mammalian serum or urine. Therefore, any physiological effects of dietary InsP6 can only be due either to its actions in the gut as a polyvalent cation chelator, or to inositol generated by its dephosphorylation by gut microflora.

A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine.

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.

Exploring phosphatidylinositol 5-phosphate 4-kinase function.

The family of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) is emerging from a comparative backwater in inositide signalling into the mainstream, as is their substrate, phosphatidylinositol 5-phosphate (PI5P). Here we review some of the key questions about the PI5P4Ks, their localisation, interaction, and regulation and also we summarise our current understanding of how PI5P is synthesised and what its cellular functions might be. Finally, some of the evidence for the involvement of PI5P4Ks in pathology is discussed.

Direct Visualisation of Abnormal Dendritic Spine Morphology in the Hippocampus of the R6/2 Transgenic Mouse Model of Huntington's Disease.

BACKGROUND: Spatial learning deficits are observed in R6/2 mice, a transgenic mouse model of Huntington's Disease (HD). Spatial learning is a hippocampal-dependent process, and impairment of memory is thought to be due, at least in part, to structural changes such as loss of dendritic spines. OBJECTIVE: To analyse dendritic spines in the hippocampus of R6/2 mice to determine if there are changes that correlate with the hippocampal dysfunction observed in these mice. METHODS: A double transgenic cross between R6/2 mice and a reporter line (YFP-H) of mice that express yellow fluorescent protein (YFP) in a subset of their neurons was used. This allowed us to visualise dendritic spines in the brains of R6/2 mice directly. RESULTS: Clear differences were seen in the distribution of YFP in the hippocampal formation of wild-type (WT)-YFP-H and R6/2-YFP-H mice, particularly in the CA1 region. We quantified dendritic spine density and dendritic spine length in the apical dendrites of the CA1 hippocampal neurons. A significant reduction in dendritic spine density, and a concomitant increase in dendritic spine length was observed in R6/2-YFP-H mice compared to WT-YFP-H mice. CONCLUSION: The R6/2-YFP-H mouse is a useful tool for directly visualising dendritic spines in the brain of a Huntington's disease mouse model. The changes we observed in dendritic spine density and length in the hippocampus might contribute to the synaptic plasticity deficits and behavioural alteration of impaired spatial learning seen in R6/2 mice.