Potent Sensitisation of Cancer Cells to Anticancer Drugs by a Quadruple Mutant of the Human Deoxycytidine Kinase.

Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK) mutant (G12) that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36) that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC), for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Potent sensitisation of cancer cells to anticancer drugs by a quadruple mutant of the human deoxycytidine kinase

Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK) mutant (G12) that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36) that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC), for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches

Scheme of the procedure followed to generate F27-RL and F11-DL.

<p>The general structure of the genomic RNAs used for retrovolution is given at the top, on the left for the F16 population and on the right for the population containing only the G12 variant. The procedure has been previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140741#pone.0140741.ref034" target="_blank">34</a>] and consists in the repeated transduction of HEK 293T cells with the lentiviral vectors followed by the selection, with puromycin, of cell populations that have stably integrated the proviral DNA that has been produced by reverse transcription. Transfection of this population with the transcomplementation plasmids pHCMV-G and pCMV∆8.91 (see main text) allows the production of the next vector generation that is then used to transduce fresh HEK 293T cells during repeated cycles of selection, transfection and transduction. For screening purposes, target cells are transduced by less than one lentivector particle per cell, and individual clones were isolated in the presence of puromycin. Screening for sensitisation to various compounds is carried out as described in the main text.</p

The M36 mutant.

<p>Panel A. Sensitivity to gemcitabine of Messa10K cells induced by M36. The ratio of living cells over the total number of cells is plotted as a function of the concentration of gemcitabine. Data are the average of five independent experiments. Panel B. Localisation of the mutations in the human dCK that characterise M36. The nucleotide sequence (double stranded) is given above, with the aminoacid sequence below. The full-length protein is given by a pale blue bar with the major functional domains, as defined in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140741#pone.0140741.ref028" target="_blank">28</a>], indicated in colour.</p

Contribution of the individual mutations of G12 to the observed phenotype.

<p>Panel A. Structure of the genomic RNA generated by transcription after transfection of cells with pSDY-SIN plasmids. R, repeated sequence from HIV-1 genome; U5, 5' unique sequence of HIV-1 LTR; Cis-acting, sequences required for packaging and reverse transcription of the genomic RNA; EF1-alpha, human elongation factor 1-alpha promoter; hPGK, human phosphoglycerate kinase promoter; Puro, puromycin N-acetyl-transferase gene; ∆U3, partially deleted version of the 3' unique sequence of HIV-1 LTR. Panels B and C. The proportion of alive cells over the total number of cells as function of the concentration in gemcitabine is given as a ratio with respect to the alive cells observed in the absence of drug. White symbols represent reference populations in both panels and. grey symbols represent single and double mutants (as described in the main text). Since single and double mutants were tested in parallel in the same experiment. The data are the average of 3 independent experiments. Error bars are not shown for the sake of clarity.</p

Biochemical comparative characterisation of phosphorylation of AraC by wt dCK and M36.

<p>Panels A and C. Phosphorylation kinetics of purified wt dCK (black squares) and M36 (red circles). Steady state kinetic data were fitted according to the Michaelis-Menten equation. Panel A, phosphorylation of dC (average of four independent experiments). Panel B, phosphorylation of gemcitabine (average of four independent experiments). Panel C, phosphorylation of AraC (average of three independent experiments). Panel D, ratio of Km (black) and of Kcat (grey) for M36 vs G12, with respect to dC and to gemcitabine. The dotted line gives the reference of a ratio of 1.</p

Position of the mutations found in the individual clones.

<p>In each panel, the dCK protein is represented (top of the drawing) as a pale blue box with the main structural motifs given in color (the first and last aminoacid of each motif is given below the box): purple, P-loop; yellow, insert; green, ERS; blue, lid; pink, base sensing loop; as defined in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140741#pone.0140741.ref028" target="_blank">28</a>]. For each panel, a cumulative map of the positions that were mutated is provided at the top of the box with aminoacid substitutions given in red, while aminoacids that did not change but with associated codons that carried synonymous mutations given in black. Panels A and B respectively show the patterns observed for the random and directed libraries, respectively. Two deletion were also found: one single nucleotide deletion in clone 6 indicated as "193 out of frame", and a two-nucleotide deletion (positions 141–142) in clone 36 indicated by a red horizontal line. In Panel B, clone 36 (corresponding to M36 in the main text, for Mutant 36) is given in bold.</p

Use of a self-inactivating (SIN) LTR sequence.

<p>Panel A, sensitivity of cells harbouring a proviral DNA carrying SIN LTR and expressing M36 (white circles); of cells harbouring a proviral DNA carrying wt LTR and expressing M36 (grey circles, the data for this curve are reported as reference from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140741#pone.0140741.g004" target="_blank">Fig 4A</a>, where they appear as white diamonds); of cells harbouring a proviral DNA carrying SIN LTR and expressing the wt dCK (white triangles); of wt Messa 10K cells (white squares). The proportion of living cells over the total number of cells is plotted as a function of the concentration of gemcitabine. The averages of three independent experiments are presented. Panel B, Western blot analysis of the expression of wt dCK and of M36 in wt-LTR and in SIN-LTR vectors. The band corresponding to the human dCK is shown for protein extracts from a population of Messa 10K cells expressing the M36 variant from a proviral DNA containing either a wt LTR or a SIN LTR. Three different amounts of total protein extract were loaded for each sample, as indicated (in μg) above each lane. The picture shows a representative result of 3 independent Western blot experiments.</p

Mutation panel found in the dCK coding-sequence in F27-RL and in F10-DL.

<p>Frequencies of mutation are calculated as described in Materials and Methods; %G>A mutations: % of transitions from G to A among the number of mutated positions found; mutations/clone: range of mutations found in single clones (for the F10-DL library the three mutations present in G12 are not considered).</p><p>Mutation panel found in the dCK coding-sequence in F27-RL and in F10-DL.</p