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Microtubule poisons are microtubule targeting agents widely used in cancer treatment. These drugs are given to cancer patients in cycles, and resistant cells can emerge both after the first or the following rounds of treatment, enhancing tumor relapse. Microtubule poisons arrest cell division by the activation of the Spindle Assembly Checkpoint (SAC). The latter is a signaling pathway that leads to the inhibition of Cdc20, the activator of the Anaphase Promoting Complex/Cyclosome (APC/C), the E3 ligase ultimately responsible for anaphase onset. Very often, Cdc20 is overexpressed in cancer cells. Surprisingly, however these cells are checkpoint proficient, although they slip out from the arrest more easily than wild type cells. When cells are treated with microtubule poisons, such as nocodazole, proper Kinetochore-Microtubule attachments are prevented, causing in a fraction of cells constant SAC signaling, prolonged arrest in prometaphase and cell death. However, a subpopulation of cells can overcome the arrest with a process called “mitotic slippage”. As a consequence, cells divide even when chromosome segregation is impaired. Aneuploidy arises as a consequence. In the current thesis, I analyzed how yeast and human cells become prone to survive when microtubule polymerization is hindered. Taking advantage of mutations in beta-tubulin, we allowed 24 clonal populations of yeast cells unable to properly polymerize microtubules to evolve in the lab for ~150 generations. At the end of the evolution experiment, cells had re-gained the ability to form microtubules, and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing allowed us to identify recurrently-mutated genes, in particular for tubulins and kinesins, as well as the widespread duplication of chromosome VIII. Recapitulating these mutations and disomy of chromosome VIII prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Analysis of the temporal order of mutations leading to resistance in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by one, and only one, additional adaptive mutation in either tubulins or kinesins. In the same way, we evolved yeast cells overexpressing CDC20 unable to properly polymerize microtubules. We found that CDC20 overexpressing cells were able to develop resistance faster than cells with physiological CDC20 levels. They did it following a different evolutionary trajectory: first they acquired Chromosome VIII disomy then they mostly mutated genes involved in Non-Sense mediated RNA decay pathway (NMD). We showed that NMD mutations are adaptive and do not tend to occur in cells with physiological CDC20 expression. We then explored the consequences of prolonged inactivation of microtubules in mammalian cells. During the mitotic arrest, human cells, experience a partial activation of the apoptotic process, which is guided by an incomplete Mitochondria Outer Membrane Permeabilization and subsequent Caspase3/7 activation. This results in DNA damage followed by p53 activation after mitotic slippage. We analyzed hTERT-RPE1 cells that slip out form mitosis for the first time (FC-First Cycle) from a mitotic arrest induced by low doses of nocodazole (i.e. microtubule poisons) and cells that slip-out from mitosis for the second time (SC-Second Cycle). We found that both populations restore growth after drug removal. However, SC cells restore their growth faster than FC cells. Even if the timing of mitotic arrest is comparable in FC and SC cells in the presence of the drug, SC cells tend to die less and are more likely to slip-out of mitosis. In addition, we discovered an increase in the expression of proteins with anti-apoptotic properties by proteomic analysis on the growing populations of FC and SC cells. Among these proteins, we focused on Triap1 whose overexpression causes minor caspase3/7 activation and DNA damage accumulation during nocodazole treatment, thereby improving cell recovery after drug removal. In addition, overexpression of Triap1 allows FC cells to behave like SC cells expressing endogenous Triap1 levels, while Triap1 downregulation forces SC cells to behave like FC cells. Our results prove that Triap1 overexpression desensitizes cells to nocodazole treatment. 2025-01-25

Intracellular reaction rates depend on concentrations and hence their levels are often regulated. However classical models of stochastic gene expression lack a cell size description and cannot be used to predict noise in concentrations. Here, we construct a model of gene product dynamics that includes a description of cell growth, cell division, size-dependent gene expression, gene dosage compensation, and size control mechanisms that can vary with the cell cycle phase. We obtain expressions for the approximate distributions and power spectra of concentration fluctuations which lead to insight into the emergence of concentration homeostasis. We find that (i) the conditions necessary to suppress cell division-induced concentration oscillations are difficult to achieve; (ii) mRNA concentration and number distributions can have different number of modes; (iii) two-layer size control strategies such as sizer-timer or adder-timer are ideal because they maintain constant mean concentrations whilst minimising concentration noise; (iv) accurate concentration homeostasis requires a fine tuning of dosage compensation, replication timing, and size-dependent gene expression; (v) deviations from perfect concentration homeostasis show up as deviations of the concentration distribution from a gamma distribution. Some of these predictions are confirmed using data for E. coli, fission yeast, and budding yeast.