Interplay of mutations, alternate mechanisms, and treatment breaks in leukaemia: Understanding and implications studied with stochastic models.

Bcr-Abl1 kinase domain mutations are the most prevalent cause of treatment resistance in chronic myeloid leukaemia (CML). Alternate resistance pathways nevertheless exist, and cell line experiments show certain patterns in the gain, and loss, of some of these alternate adaptations. These adaptations have clinical consequences when the tumour develops mechanisms that are beneficial to its growth under treatment, but slow down its growth when not treated. The results of temporarily halting treatment in CML have not been widely discussed in the clinic and there is no robust theoretical model that could suggest when such a pause in therapy can be tolerated. We constructed a dynamic model of how mechanisms such as Bcr-Abl1 overexpression and drug transporter upregulation evolve to produce resistance in cell lines, and investigate its behaviour subject to different treatment schedules, in particular when the treatment is paused ('drug holiday'). Our study results suggest that the presence of additional resistance mechanisms creates an environment which favours mutations that are either preexisting or occur late during treatment. Importantly, the results suggest the existence of tumour drug addiction, where cancer cells become dependent on the drug for (optimal) survival, which could be exploited through a treatment holiday. All simulation code is available at https://github.com/Sandalmoth/dual-adaptation.

Rotating between ponatinib and imatinib temporarily increases the efficacy of imatinib as shown in a chronic myeloid leukaemia model.

Targeted therapies for chronic myeloid leukaemia (CML) are effective, but rarely curative. Patients typically require treatment indefinitely, which gives ample time for drug resistance to evolve. Drug resistance issues are one of the main causes of death owing to CML, thus any means of preventing resistance are of importance. Drug rotations, wherein treatment is switched periodically between different drugs are one such option, and have been theorized to delay the onset of resistance. In vitro testing of drug rotation therapy is a first step towards applying it in animal or human trials. We developed a method for testing drug rotation protocols in CML cell lines based around culturing cells with a moderate amount of inhibitors interspersed with washing procedures and drug swaps. Drug rotations of imatinib and ponatinib were evaluated in a CML specific cell line, KCL-22. The growth of KCL-22 cells was initially reduced by a drug rotation, but the cells eventually adapted to the protocol. Our results show that ponatinib in a drug rotation temporarily sensitizes the cells to imatinib, but the effect is short-lived and is eventually lost after a few treatment cycles. Possible explanations for this observation are discussed.

Combating drug resistance in acute myeloid leukaemia by drug rotations: the effects of quizartinib and pexidartinib.

BACKGROUND: Acute myeloid leukaemia (AML) is an aggressive blood cancer. In approximately 30% of the cases, driver mutations in the FLT3 gene are identified. FLT3 inhibitors are used in treatment of such patients together with cytotoxic drugs or (in refractory AML) as single agents. Unfortunately, resistance to FLT3 inhibitors limits their efficacy. Resistance is often due to secondary mutations in the gene encoding the molecular target. The gatekeeper mutation F691L confers resistance to specific FLT3 inhibitors such as quizartinib, but pexidartinib is much less resistance to this mutation. Pexidartinib alone is however sensitive to many other resistance mutations. In chronic myeloid leukaemia (CML), it has been suggested that rotation between drugs with a different landscape of resistance mutations might postpone the emergence of resistance. METHODS: We studied the effect of quizartinib and pexidartinib in AML cell lines that express FLT3 (MOLM-14 and MV4-11). Using a rotation protocol, we further examined whether the emergence of resistance could be postponed. Computational modelling was used to analyse the onset of resistance and suggest which mutations are most likely to occur in a quantitative fashion. RESULTS: The cells were sensitive to both inhibitors but quickly developed resistance that could be inherited, suggesting a genetic origin. Rotation protocols were not useful to postpone the emergence of resistance, which implies that such protocols, or changing from pexidartinib to quizartinib (or vice-versa) should not be used in patients. The computational modelling led to similar conclusions and suggested that F691L is the most common mutation to occur with quizartinib, and also when both drugs are used in rotation. CONCLUSIONS: AML patients are not likely to benefit from a quizartinib/pexidartinib rotation protocol. A combination of tyrosine kinase inhibitors (with different molecular targets) might be more useful in the future. Development of specific FLT3 inhibitors that are less sensitive to resistance mutations might also lead to a better outcome.

Inferring time-dependent population growth rates in cell cultures undergoing adaptation.

BACKGROUND: The population growth rate is an important characteristic of any cell culture. During sustained experiments, the growth rate may vary due to competition or adaptation. For instance, in presence of a toxin or a drug, an increasing growth rate indicates that the cells adapt and become resistant. Consequently, time-dependent growth rates are fundamental to follow on the adaptation of cells to a changing evolutionary landscape. However, as there are no tools to calculate the time-dependent growth rate directly by cell counting, it is common to use only end point measurements of growth rather than tracking the growth rate continuously. RESULTS: We present a computer program for inferring the growth rate over time in suspension cells using nothing but cell counts, which can be measured non-destructively. The program was tested on simulated and experimental data. Changes were observed in the initial and absolute growth rates, betraying resistance and adaptation. CONCLUSIONS: For experiments where adaptation is expected to occur over a longer time, our method provides a means of tracking growth rates using data that is normally collected anyhow for monitoring purposes. The program and its documentation are freely available at https://github.com/Sandalmoth/ratrack under the permissive zlib license.

Stochastic modelling of tyrosine kinase inhibitor rotation therapy in chronic myeloid leukaemia.

BACKGROUND: Resistance towards targeted cancer treatments caused by single nucleotide variations is a major issue in many malignancies. Currently, there are a number of available drugs for chronic myeloid leukaemia (CML), which are overcome by different sets of mutations. The main aim of this study was to explore if it can be possible to exploit this and create a treatment protocol that outperforms each drug on its own. METHODS: We present a computer program to test different treatment protocols against CML, based on available resistance mutation growth data. The evolution of a relatively stable pool of cancer stem cells is modelled as a stochastic process, with the growth of cells expressing a tumourigenic protein (here, Abl1) and any emerging mutants determined principally by the drugs used in the therapy. RESULTS: There can be some benefit to Bosutinib-Ponatinib rotation therapy even if the mutation status is unknown, whereas Imatinib-Nilotinib rotation is unlikely to improve the outcomes. Furthermore, an interplay between growth inhibition and selection effects generates a non-linear relationship between drug doses and the risk of developing resistance. CONCLUSIONS: Drug rotation therapy might be able to delay the onset of resistance in CML patients without costly ongoing observation of mutation status. Moreover, the simulations give credence to the suggestion that lower drug concentrations may achieve better results following major molecular response in CML.