FUNCTIONAL AND GENETIC HETEROGENEITY IN ACUTE MYELOID LEUKAEMIA

Acute myeloid leukaemia (AML) is the most frequent leukaemia in adults, and still represents a disease with an unmet medical need, with 50-60% of patients relapsing within 3 years after diagnosis. AMLs are characterised by a high degree of intra-tumour heterogeneity, both at the biological and the genetic level, which is critical for tumour maintenance and response to treatments. Biologically, AMLs are organised hierarchically, with rare stem-like cells (leukaemia stem cells, LSCs) endowed with the unique properties of self-renewal and differentiation. Genetically, AMLs harbour patient-specific combinations of different driver mutations, which are organised within individual cases in sub-clones with distinct growth properties. We hypothesized that tumour maintenance and relapse in AMLs are driven by the selective expansion of quiescent sub-clones within the LSC population, which serve as the genomic and functional reservoir of the tumour. The experimental strategy we employed to test this hypothesis is based on the xenotransplantation of human leukaemias, the implementation of an in vivo clonal tracking approach, the functional isolation of leukaemic subpopulations with diverse proliferation histories and whole-exome sequencing (WES) of bulk and isolated leukaemic subpopulations. Our aims were to assess the proliferative hierarchy of LSCs and to examine their intrinsic genetic heterogeneity. We identified two functional LSC classes, quiescent and cycling, that are in equilibrium in the tumour and largely share the same clonal architecture. We further observed that genetic leukaemic clones appear to consist of a high number of individual LSCs, the majority of which exhaust upon serial transplantation. Finally, by genetic analyses of isolated leukaemic subsets, we were able to detect a specific enrichment for rare mutations in the quiescent compartment of two patient xenografts. Our data indicate that tumour evolution is sustained by the quiescent LSC pool and suggest that their highly proliferating counterpart has a finite lifespan. We expect that the results of our studies will provide new insights into the mechanisms of disease progression and treatment response in AML, and potentially reveal novel therapeutic approaches

Modeling cell proliferation in human acute myeloid leukemia xenografts

Motivation: Acute myeloid leukemia (AML) is one of the most common hematological malignancies, characterized by high relapse and mortality rates. The inherent intra-tumor heterogeneity in AML is thought to play an important role in disease recurrence and resistance to chemotherapy. Although experimental protocols for cell proliferation studies are well established and widespread, they are not easily applicable to in vivo contexts, and the analysis of related time-series data is often complex to achieve. To overcome these limitations, model-driven approaches can be exploited to investigate different aspects of cell population dynamics. Results: In this work, we present ProCell, a novel modeling and simulation framework to investigate cell proliferation dynamics that, differently from other approaches, takes into account the inherent stochasticity of cell division events. We apply ProCell to compare different models of cell proliferation in AML, notably leveraging experimental data derived from human xenografts in mice. ProCell is coupled with Fuzzy Self-Tuning Particle Swarm Optimization, a swarm-intelligence settings-free algorithm used to automatically infer the models parameterizations. Our results provide new insights on the intricate organization of AML cells with highly heterogeneous proliferative potential, highlighting the important role played by quiescent cells and proliferating cells characterized by different rates of division in the progression and evolution of the disease, thus hinting at the necessity to further characterize tumor cell subpopulations. Availability and implementation: The source code of ProCell and the experimental data used in this work are available under the GPL 2.0 license on GITHUB at the following URL: https://github.com/aresio/ProCell

Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites

Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic analysis if it is to be of use as a molecular genetic tool for malaria parasites

Spontaneous regression of metastatic renal cell carcinoma: case report

Spontaneous regression of metastatic renal cell carcinoma is rarely observed. A case of suspected spontaneous regression of pulmonary metastases following nephrectomy for histologically proven renal cell carcinoma without systemic treatment is presented along with a brief review of the literature

Microbial adaptation to venom is common in snakes and spiders

Animal venoms are considered sterile sources of antimicrobial compounds with strong membrane disrupting activity against multi-drug resistant bacteria. However, bite wound infections are common in developing nations. Investigating the oral and venom microbiome of five snake and two spider species, we evidence viable microorganisms potentially unique to venom for black-necked spitting cobras (Naja nigricollis). Among these are two novel sequence types of Enterococcus faecalis misidentified by commonly used clinical biochemistry procedures as Staphylococcus; the genome sequence data of venom-specific isolates feature an additional 45 genes, at least 11 of which improve membrane integrity. Our findings challenge the dogma of venom sterility and indicate an increased primary infection risk in the clinical management of venomous animal bite wounds

Anopheles Gambiae PRS1 Modulates Plasmodium Development at Both Midgut and Salivary Gland Steps

Background: Invasion of the mosquito salivary glands by Plasmodium is a critical step for malaria transmission. From a SAGE analysis, we previously identified several genes whose expression in salivary glands was regulated coincident with sporozoite invasion of salivary glands. To get insights into the consequences of these salivary gland responses, here we have studied one of the genes, PRS1 (Plasmodium responsive salivary 1), whose expression was upregulated in infected glands, using immunolocalization and functional inactivation approaches. Methodology/Principal Findings: PRS1 belongs to a novel insect superfamily of genes encoding proteins with DM9 repeat motifs of uncharacterized function. We show that PRS1 is induced in response to Plasmodium, not only in the salivary glands but also in the midgut, the other epithelial barrier that Plasmodium has to cross to develop in the mosquito. Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum. In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells. We also find that sporozoite invasion of salivary gland cells occurs sequentially and induces intra-cellular modifications that include an increase in PRS1 expression and a relocalization of the corresponding protein into vesicle-like structures. Importantly, PRS1 knockdown during the onset of midgut and salivary gland invasion demonstrates that PRS1 acts as an agonist for the development of both parasite species in the two epithelia, highlighting shared vector/parasite interactions in both tissues. Conclusions/Significance: While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history

Conserved Mosquito/Parasite Interactions Affect Development of Plasmodium falciparum in Africa

In much of sub-Saharan Africa, the mosquito Anopheles gambiae is the main vector of the major human malaria parasite, Plasmodium falciparum. Convenient laboratory studies have identified mosquito genes that affect positively or negatively the developmental cycle of the model rodent parasite, P. berghei. Here, we use transcription profiling and reverse genetics to explore whether five disparate mosquito gene regulators of P. berghei development are also pertinent to A. gambiae/P. falciparum interactions in semi-natural conditions, using field isolates of this parasite and geographically related mosquitoes. We detected broadly similar albeit not identical transcriptional responses of these genes to the two parasite species. Gene silencing established that two genes affect similarly both parasites: infections are hindered by the intracellular local activator of actin cytoskeleton dynamics, WASP, but promoted by the hemolymph lipid transporter, ApoII/I. Since P. berghei is not a natural parasite of A. gambiae, these data suggest that the effects of these genes have not been drastically altered by constant interaction and co-evolution of A. gambiae and P. falciparum; this conclusion allowed us to investigate further the mode of action of these two genes in the laboratory model system using a suite of genetic tools and infection assays. We showed that both genes act at the level of midgut invasion during the parasite's developmental transition from ookinete to oocyst. ApoII/I also affects the early stages of oocyst development. These are the first mosquito genes whose significant effects on P. falciparum field isolates have been established by direct experimentation. Importantly, they validate for semi-field human malaria transmission the concept of parasite antagonists and agonists

Chloroquine Mediated Modulation of Anopheles gambiae Gene Expression

Plasmodium development in the mosquito is crucial for malaria transmission and depends on the parasite's interaction with a variety of cell types and specific mosquito factors that have both positive and negative effects on infection. Whereas the defensive response of the mosquito contributes to a decrease in parasite numbers during these stages, some components of the blood meal are known to favor infection, potentiating the risk of increased transmission. The presence of the antimalarial drug chloroquine in the mosquito's blood meal has been associated with an increase in Plasmodium infectivity for the mosquito, which is possibly caused by chloroquine interfering with the capacity of the mosquito to defend against the infection.In this study, we report a detailed survey of the Anopheles gambiae genes that are differentially regulated by the presence of chloroquine in the blood meal, using an A. gambiae cDNA microarray. The effect of chloroquine on transcript abundance was evaluated separately for non-infected and Plasmodium berghei-infected mosquitoes. Chloroquine was found to affect the abundance of transcripts that encode proteins involved in a variety of processes, including immunity, apoptosis, cytoskeleton and the response to oxidative stress. This pattern of differential gene expression may explain the weakened mosquito defense response which accounts for the increased infectivity observed in chloroquine-treated mosquitoes.The results of the present study suggest that chloroquine can interfere with several putative mosquito mechanisms of defense against Plasmodium at the level of gene expression and highlight the need for a better understanding of the impacts of antimalarial agents on parasite transmission