Oncolytic Virotherapy for Hematological Malignancies

Hematological malignancies such as leukemias, lymphomas, multiple myeloma (MM), and the myelodysplastic syndromes (MDSs) primarily affect adults and are difficult to treat. For high-risk disease, hematopoietic stem cell transplant (HCT) can be used. However, in the setting of autologous HCT, relapse due to contamination of the autograft with cancer cells remains a major challenge. Ex vivo manipulations of the autograft to purge cancer cells using chemotherapies and toxins have been attempted. Because these past strategies lack specificity for malignant cells and often impair the normal hematopoietic stem and progenitor cells, prior efforts to ex vivo purge autografts have resulted in prolonged cytopenias and graft failure. The ideal ex vivo purging agent would selectively target the contaminating cancer cells while spare normal stem and progenitor cells and would be applied quickly without toxicities to the recipient. One agent which meets these criteria is oncolytic viruses. This paper details experimental progress with reovirus, myxoma virus, measles virus, vesicular stomatitis virus, coxsackievirus, and vaccinia virus as well as requirements for translation of these results to the clinic

TRP channels as potential targets for antischistosomals

Ion channels are membrane protein complexes that underlie electrical excitability in cells, allowing ions to diffuse through cell membranes in a regulated fashion. They are essential for normal functioning of the neuromusculature and other tissues. Ion channels are also validated targets for many current anthelmintics, yet the properties of only a small subset of ion channels in parasitic helminths have been explored in any detail. Transient receptor potential (TRP) channels comprise a widely diverse superfamily of ion channels with important roles in sensory signaling, regulation of ion homeostasis, organellar trafficking, and other functions. There are several subtypes of TRP channels, including TRPA1 and TRPV1 channels, both of which are involved in, among other functions, sensory, nociceptive, and inflammatory signaling in mammals. Several lines of evidence indicate that TRPA1-like channels in schistosomes exhibit pharmacological sensitivities that differ from their mammalian counterparts and that may signify unique physiological properties as well. Thus, in addition to responding to TRPA1 modulators, schistosome TRPA1-like channels also respond to compounds that in other organisms modulate TRPV1 channels. Notably, TRPV channel genes are not found in schistosome genomes. Here, we review the evidence leading to these conclusions and examine potential implications. We also discuss recent results showing that praziquantel, the current drug of choice against schistosomiasis, selectively targets host TRP channels in addition to its likely primary targets in the parasite. The results we discuss add weight to the notion that schistosome TRP channels are worthy of investigation as candidate therapeutic targets. Keywords: Schistosoma, Schistosomiasis, Ion channels, TRP channels, Capsaicin, TRPA1, TRPV1, Praziquante

TRP channels in schistosomes

Praziquantel (PZQ) is effectively the only drug currently available for treatment and control of schistosomiasis, a disease affecting hundreds of millions of people worldwide. Many anthelmintics, likely including PZQ, target ion channels, membrane protein complexes essential for normal functioning of the neuromusculature and other tissues. Despite this fact, only a few classes of parasitic helminth ion channels have been assessed for their pharmacological properties or for their roles in parasite physiology. One such overlooked group of ion channels is the transient receptor potential (TRP) channel superfamily. TRP channels share a common core structure, but are widely diverse in their activation mechanisms and ion selectivity. They are critical to transducing sensory signals, responding to a wide range of external stimuli. They are also involved in other functions, such as regulating intracellular calcium and organellar ion homeostasis and trafficking. Here, we review current literature on parasitic helminth TRP channels, focusing on those in schistosomes. We discuss the likely roles of these channels in sensory and locomotor activity, including the possible significance of a class of TRP channels (TRPV) that is absent in schistosomes. We also focus on evidence indicating that at least one schistosome TRP channel (SmTRPA) has atypical, TRPV1-like pharmacological sensitivities that could potentially be exploited for future therapeutic targeting

Epstein-Barr Virus SM Protein Utilizes Cellular Splicing Factor SRp20 To Mediate Alternative Splicing▿

Epstein-Barr virus (EBV) SM protein is an essential nuclear protein produced during the lytic cycle of EBV replication. SM is an RNA-binding protein with multiple mechanisms of action. SM enhances the expression of EBV genes by stabilizing mRNA and facilitating nuclear export. SM also influences splicing of both EBV and cellular pre-mRNAs. SM modulates splice site selection of the host cell STAT1 pre-mRNA, directing utilization of a novel 5′ splice site that is used only in the presence of SM. SM activates splicing in the manner of SR proteins but does not contain the canonical RS domains typical of cellular splicing factors. Affinity purification and mass spectrometry of SM complexes from SM-transfected cells led to the identification of the cellular SR splicing factor SRp20 as an SM-interacting protein. The regions of SM and SRp20 required for interaction were mapped by in vitro and in vivo assays. The SRp20 interaction was shown to be important for the effects of SM on alternative splicing by the use of STAT1 splicing assays. Overexpression of SRp20 enhanced SM-mediated alternative splicing and knockdown of SRp20 inhibited the SM effect on splicing. These data suggest a model whereby SM, a viral protein, recruits and co-opts the function of cellular SRp20 in alternative splicing

Evidence for Novel Pharmacological Sensitivities of Transient Receptor Potential (TRP) Channels in <i>Schistosoma mansoni</i>

<div><p>Schistosomiasis, caused by parasitic flatworms of the genus <i>Schistosoma</i>, is a neglected tropical disease affecting hundreds of millions globally. Praziquantel (PZQ), the only drug currently available for treatment and control, is largely ineffective against juvenile worms, and reports of PZQ resistance lend added urgency to the need for development of new therapeutics. Ion channels, which underlie electrical excitability in cells, are validated targets for many current anthelmintics. Transient receptor potential (TRP) channels are a large family of non-selective cation channels. TRP channels play key roles in sensory transduction and other critical functions, yet the properties of these channels have remained essentially unexplored in parasitic helminths. TRP channels fall into several (7–8) subfamilies, including TRPA and TRPV. Though schistosomes contain genes predicted to encode representatives of most of the TRP channel subfamilies, they do not appear to have genes for any TRPV channels. Nonetheless, we find that the TRPV1-selective activators capsaicin and resiniferatoxin (RTX) induce dramatic hyperactivity in adult worms; capsaicin also increases motility in schistosomula. SB 366719, a highly-selective TRPV1 antagonist, blocks the capsaicin-induced hyperactivity in adults. Mammalian TRPA1 is not activated by capsaicin, yet knockdown of the single predicted TRPA1-like gene (SmTRPA) in <i>S</i>. <i>mansoni</i> effectively abolishes capsaicin-induced responses in adult worms, suggesting that SmTRPA is required for capsaicin sensitivity in these parasites. Based on these results, we hypothesize that some schistosome TRP channels have novel pharmacological sensitivities that can be targeted to disrupt normal parasite neuromuscular function. These results also have implications for understanding the phylogeny of metazoan TRP channels and may help identify novel targets for new or repurposed therapeutics.</p></div

Knockdown of SmTRPA attenuates the hyperactivity elicited by the TRPA1 activator AITC.

<p>Adult males (<b>A</b>) and females (<b>B</b>) were exposed to different concentrations of AITC, and motility measured, as described in Figs <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004295#pntd.0004295.g001" target="_blank">1</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004295#pntd.0004295.g004" target="_blank">4</a>. The AITC-dependent normalized change in motility at each AITC concentration for these control worms (not exposed to any siRNA; 10, 20, 40, 60, 100) is compared with the AITC-dependent normalized change in motility for worms with SmTRPA expression suppressed by RNAi (10 KD, 20 KD, 40 KD, 60 KD, 100 KD). *, ***, ****, P<0.05, P<0.001, P<0.0001 respectively, unpaired t-test. n = 4–8.</p

SB 366791, a potent, highly-selective TRPV1 antagonist, inhibits capsaicin-induced increased motility in <i>S</i>. <i>mansoni</i> adults.

<p>Motility was measured as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004295#pntd.0004295.g001" target="_blank">Fig 1</a> in individual males (<b>A</b>) and females (<b>B</b>). White bars show the significant increase in worm motility in response to 10 μM capsaicin in the absence of SB 366791. Differently patterned bars designate worm motility in 0 vs. 10 μM capsaicin following pre-incubation in (and co-exposure to) SB 366791 at concentrations of 0.4 μM (horizontal lines), 0.5 μM (checkerboard), 1 μM (diagonal lines), 2 μM (squares) or 10 μM (vertical lines). The pair of black bars shows responses of worms to 100 μM capsaicin in the presence of 100 μM SB 366791 (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004295#sec002" target="_blank">Methods</a> for details). There is a significant increase in worm motility in response to capsaicin only in the absence of SB 366791. *, **, ***, P<0.05, P<0.01, P<0.001 respectively, paired t-tests vs. no-capsaicin controls. n = 4–8.</p

Knockdown of SmTRPA eliminates schistosome response to capsaicin.

<p>Comparison of capsaicin-dependent worm motility between normal adult worms (eg, no electroporation or knockdown) and those electroporated with 5 μg SmTRPA siRNA (KD). (<b>A</b>) Male worms. (<b>B</b>) Female worms. Capsaicin concentrations are shown. Those bars labeled with only the capsaicin concentration (20, 60, 100) on the X axis designate normalized motility following exposure to that concentration of capsaicin in worms that have not been subjected to knockdown. Those showing the capsaicin concentration plus "KD" (20 KD, 60 KD, 100 KD) designate capsaicin-induced normalized motility in worms subjected to knockdown of SmTRPA. Also shown are "Control" worms (no knockdown, no capsaicin) and worms electroporated with 5 μg luciferase siRNA (Luc) and exposed to 20 μM capsaicin as a control for effects of electroporation with siRNA. Worm activity was assessed as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004295#pntd.0004295.g001" target="_blank">Fig 1</a>. *, **, ***, P<0.05, P<0.01, P<0.001 respectively, unpaired t-test vs. control worms for each capsaicin concentration. n = 5–10. <b>C.</b> Knockdown of SmTRPA (SmTRPA KD) has no significant effect on response of adult males (left) or females (right) to 40 μM serotonin. "Control", "Luc" labels are the same as in A and B. n = 4–6.</p

RTX, a highly specific and potent TRPV1 activator, increases motor activity in adult schistosomes.

<p>Normalized motility of adult <i>S</i>. <i>mansoni</i> males (black bars) and females (white bars) exposed to 1–10 μM RTX was measured as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004295#pntd.0004295.g001" target="_blank">Fig 1</a>. *, **, ***, P<0.05, P<0.01, P<0.001 respectively, paired t-test vs. Controls (prior to normalization). All data are presented as means ± SEM, n = 5–14 for males, 4–6 for females.</p

Examination of SmTRPA sequence for residues identified as important for drug activity.

<p>Alignments were made with ClustalW2 (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>) and Swiss-Model (<a href="http://swissmodel.expasy.org/" target="_blank">http://swissmodel.expasy.org/</a>). Numbering is based on the SmTRPA amino acid sequence. SmTRPA residues that are conserved are marked with a check mark (✓) above and are in bold and shaded, and those that are not conserved are marked with an x. <b>A.</b> Cysteine residues identified in mouse (equivalent to SmTRPA 407, 414, 611) or human (611, 631, 654) as important for activity of AITC and other electrophilic compounds on TRPA1 are shown. A lysine residue (K694) appears to be critical in human TRPA1 as well, and is also shown in bold and shaded. <b>B.</b> Residues identified as important for activity of vanilloid compounds on TRPV1 are not well conserved in SmTRPA. Of 4 residues thought to be important for capsaicin and other vanilloid activity, only the methionine/leucine residue at SmTRPA residue L824 appears to be conserved.</p