Novel phenotype of Wolbachia strain wPip in Aedes aegypti challenges assumptions on mechanisms of Wolbachia-mediated dengue virus inhibition.

The bacterial endosymbiont Wolbachia is a biocontrol tool that inhibits the ability of the Aedes aegypti mosquito to transmit positive-sense RNA viruses such as dengue and Zika. Growing evidence indicates that when Wolbachia strains wMel or wAlbB are introduced into local mosquito populations, human dengue incidence is reduced. Despite the success of this novel intervention, we still do not fully understand how Wolbachia protects mosquitoes from viral infection. Here, we demonstrate that the Wolbachia strain wPip does not inhibit virus infection in Ae. aegypti. We have leveraged this novel finding, and a panel of Ae. aegypti lines carrying virus-inhibitory (wMel and wAlbB) and non-inhibitory (wPip) strains in a common genetic background, to rigorously test a number of hypotheses about the mechanism of Wolbachia-mediated virus inhibition. We demonstrate that, contrary to previous suggestions, there is no association between a strain's ability to inhibit dengue infection in the mosquito and either its typical density in the midgut or salivary glands, or the degree to which it elevates innate immune response pathways in the mosquito. These findings, and the experimental platform provided by this panel of genetically comparable mosquito lines, clear the way for future investigations to define how Wolbachia prevents Ae. aegypti from transmitting viruses.

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry.

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequence-specificity of four VapC proteins from two different organisms (VapC(PAE0151) and VapC(PAE2754) from Pyrobaculum aerophilum, and VapC(Rv0065) and VapC(Rv0617) from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.

Successful introgression of wMel Wolbachia into Aedes aegypti populations in Fiji, Vanuatu and Kiribati.

Pacific Island countries have experienced periodic dengue, chikungunya and Zika outbreaks for decades. The prevention and control of these mosquito-borne diseases rely heavily on control of Aedes aegypti mosquitoes, which in most settings are the primary vector. Introgression of the intracellular bacterium Wolbachia pipientis (wMel strain) into Ae. aegypti populations reduces their vector competence and consequently lowers dengue incidence in the human population. Here we describe successful area-wide deployments of wMel-infected Ae. aegypti in Suva, Lautoka, Nadi (Fiji), Port Vila (Vanuatu) and South Tarawa (Kiribati). With community support, weekly releases of wMel-infected Ae. aegypti mosquitoes for between 2 to 5 months resulted in wMel introgression in nearly all locations. Long term monitoring confirmed a high, self-sustaining prevalence of wMel infecting mosquitoes in almost all deployment areas. Measurement of public health outcomes were disrupted by the Covid19 pandemic but are expected to emerge in the coming years.

wMel Wolbachia genome remains stable after 7 years in Australian Aedes aegypti field populations.

Infection of wMel Wolbachia in Aedes aegypti imparts two signature features that enable its application for biocontrol of dengue. First, the susceptibility of mosquitoes to viruses such as dengue and Zika is reduced. Second, a reproductive manipulation is caused that enables wMel introgression into wild-type mosquito populations. The long-term success of this method relies, in part, on evolution of the wMel genome not compromising the critical features that make it an attractive biocontrol tool. This study compared the wMel Wolbachia genome at the time of initial releases and 1-7 years post-release in Cairns, Australia. Our results show the wMel genome remains highly conserved up to 7 years post-release in gene sequence, content, synteny and structure. This work suggests the wMel genome is stable in its new mosquito host and, therefore, provides reassurance on the potential for wMel to deliver long-term public-health impacts.

Assessment of fitness and vector competence of a New Caledonia wMel Aedes aegypti strain before field-release.

BACKGROUND: Biological control programs involving Wolbachia-infected Aedes aegypti are currently deployed in different epidemiological settings. New Caledonia (NC) is an ideal location for the implementation and evaluation of such a strategy as the only proven vector for dengue virus (DENV) is Ae. aegypti and dengue outbreaks frequency and severity are increasing. We report the generation of a NC Wolbachia-infected Ae. aegypti strain and the results of experiments to assess the vector competence and fitness of this strain for future implementation as a disease control strategy in Noumea, NC. METHODS/PRINCIPAL FINDINGS: The NC Wolbachia strain (NC-wMel) was obtained by backcrossing Australian AUS-wMel females with New Caledonian Wild-Type (NC-WT) males. Blocking of DENV, chikungunya (CHIKV), and Zika (ZIKV) viruses were evaluated via mosquito oral feeding experiments and intrathoracic DENV challenge. Significant reduction in infection rates were observed for NC-wMel Ae. aegypti compared to WT Ae. aegypti. No transmission was observed for NC-wMel Ae. aegypti. Maternal transmission, cytoplasmic incompatibility, fertility, fecundity, wing length, and insecticide resistance were also assessed in laboratory experiments. Ae. aegypti NC-wMel showed complete cytoplasmic incompatibility and a strong maternal transmission. Ae. aegypti NC-wMel fitness seemed to be reduced compared to NC-WT Ae. aegypti and AUS-wMel Ae. aegypti regarding fertility and fecundity. However further experiments are required to assess it accurately. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that the NC-wMel Ae. aegypti strain is a strong inhibitor of DENV, CHIKV, and ZIKV infection and prevents transmission of infectious viral particles in mosquito saliva. Furthermore, our NC-wMel Ae. aegypti strain induces reproductive cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, supporting field-releases in Noumea, NC.

Preventing chemotherapy-induced myelosuppression by repurposing the FLT3 inhibitor quizartinib.

We describe an approach to inhibit chemotherapy-induced myelosuppression. We found that short-term exposure of mice to the FLT3 inhibitor quizartinib induced the transient quiescence of multipotent progenitors (MPPs). This property of quizartinib conferred marked protection to MPPs in mice receiving fluorouracil or gemcitabine. The protection resulted in the rapid recovery of bone marrow and blood cellularity, thus preventing otherwise lethal myelosuppression. A treatment strategy involving quizartinib priming that protected wild-type bone marrow progenitors, but not leukemic cells, from fluorouracil provided a more effective treatment than conventional induction therapy in mouse models of acute myeloid leukemia. This strategy has the potential to be extended for use in other cancers where FLT3 inhibition does not adversely affect the effectiveness of chemotherapy. Thus, the addition of quizartinib to cancer treatment regimens could markedly improve cancer patient survival and quality of life.

Dasatinib promotes the activation of quiescent hematopoietic stem cells in mice.

Dasatinib is an orally available broad-spectrum tyrosine kinase inhibitor that is widely used to treat chronic myeloid leukemia. It is also in clinical trials for the treatment of other malignancies, including solid tumors. Despite its wide use, little is known of its effects on normal hematopoietic stem and progenitor cells. Here, we study wild-type mice dosed with dasatinib and find that it causes the transient induction of proliferation of quiescent hematopoietic stem cells (HSCs). This finding was unexpected given the ability of dasatinib to inhibit c-Kit signaling and promote cell cycle arrest in many cell types. The transient induction of HSC proliferation in dasatinib-dosed mice coincided with a marked induction in the expression of Sca-1 and phospho-S6. Also evident at this time was a rapid but transient loss of lineage-committed hematopoietic progenitors that express high levels of c-Kit and the induction of stem cell factor in the serum. These findings suggest that activation of quiescent HSCs is part of a rapid rescue response that restores hematopoietic progenitors to pretreatment levels. This restoration coincides with HSCs returning to quiescence, and the expression of Sca-1 and phospho-S6 reverting to pre-treatment levels, even though dasatinib dosing is maintained. These data suggest that equilibrium is reached between the opposing forces of dasatinib and hematopoietic growth factors. The transient induction of HSC proliferation provided a window of opportunity whereby these cells became sensitive to killing by the cytotoxic drug 5-fluorouracil.

The targeting of human and mouse B lymphocytes by dasatinib.

Dasatinib inhibits B-cell receptor-Abelson murine leukemia viral oncogene homologue 1, Src, and other tyrosine kinases. Few studies have addressed the impact of dasatinib on normal blood cells, especially in vivo. Here we show that dasatinib leads to a reduced number of human CD19+ peripheral B cells owing to a strong induction of apoptosis. In contrast, no similar effect on T-cell viability was observed. However, dasatinib induced a comparable broad inhibition of the early events of B- and T-cell receptor signaling. Furthermore, dasatinib was shown to be a more pronounced inhibitor of both basal and B-cell receptor-induced activity of Bruton's tyrosine kinase and PLCγ2 compared with the more specific Bruton's tyrosine kinase inhibitor ibrutinib. Human progenitor B cells from the pre-B stage were sensitive to dasatinib. In an in vivo murine model, dasatinib reduced B-lineage cells in the bone marrow with a marked effect on the pre-B subpopulation. Dasatinib led to a reduced spleen size, with a loss of large immature transitional immunoglobulin M(+)/immunoglobulin D(-) B cells and a reduction in germinal center B cells. Dasatinib caused a marked loss of thymocytes without affecting myeloid lineage cells or hematopoietic progenitors. This study reveals important side effects of dasatinib with specific loss of activated B and thymocyte populations, which may have an impact during long-term treatment.

Loss of c-Cbl E3 ubiquitin ligase activity enhances the development of myeloid leukemia in FLT3-ITD mutant mice.

Mutations in the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase (RTK) occur frequently in acute myeloid leukemia (AML), with the most common involving internal tandem duplication (ITD) within the juxtamembrane domain. Fms-like tyrosine kinase 3-ITD mutations result in a mislocalized and constitutively activated receptor, which aberrantly phosphorylates signal transducer and activator of transcription 5 (STAT5) and upregulates the expression of its target genes. c-Cbl is an E3 ubiquitin ligase that negatively regulates RTKs, including FLT3, but whether it can downregulate mislocalized FLT3-ITD remains to be resolved. To help clarify this, we combined a FLT3-ITD mutation with a loss-of-function mutation in the RING finger domain of c-Cbl that abolishes its E3 ligase activity. Mice transplanted with hematopoietic stem cells expressing both mutations rapidly develop myeloid leukemia, indicating strong cooperation between the two. Although the c-Cbl mutation was shown to cause hyperactivation of another RTK, c-Kit, it had no effect on enhancing FLT3-ITD protein levels or STAT5 activation. This indicates that c-Cbl does not downregulate FLT3-ITD and that the leukemia is driven by independent pathways involving FLT3-ITD's activation of STAT5 and mutant c-Cbl's activation of other RTKs, such as c-Kit. This study highlights the importance of c-Cbl's negative regulation of wild-type RTKs in suppressing FLT3-ITD-driven myeloid leukemia.

Dasatinib targets B-lineage cells but does not provide an effective therapy for myeloproliferative disease in c-Cbl RING finger mutant mice.

This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL.