title: An investigation of a novel cyclin-dependent kinase inhibitor as a treatment option for Acute Myeloid Leukaemia
Creators: name: Chantkran, Wittawat
Publisher: name: University of Glasgow
Year: 2020
Subjects: subject: CYC065. CDK2/9 inhibitor, AML therapy, combination studies.

description: Acute myeloid leukaemia (AML) is one of the most common haematological malignancies, characterised by the clonal expansion of abnormal or poorly differentiated myeloid cells, infiltrating the bone marrow (BM), blood or extramedullary tissues. Although treatment of AML has progressed over the past few decades, in some patients, particularly elderly patients, usually defined as older than 60-65 years of age, the outcome is dismal due to the remarkable genetic complexity, epigenetic alterations, and the dynamics of the disease. In this study, a newly developed CDK2/9 inhibitor CYC065 was investigated as a treatment option for AML. OCI-AML3, MOLM-13, and MV4-11 AML cell lines were used, aiming to encompass the stratifications of major risk profiles according to the European LeukemiaNet (ELN) recommendations, to assess the effects of CYC065 on growth and survival of AML cells. It was observed that AML cells were sensitive to the treatment with CYC065. The longer CYC065 exposure, the greater the cytotoxic/anti-metabolic effect on the cells, with a reduction of the IC50. This was consistent with apoptosis results, which were induced over the 72h investigated. The percentage of active caspase-3-positive cells was also increased and correlated with an increase in the percentage of annexin V-positive cells across higher CYC065 concentrations. In cell cycle analysis, Gap 1 (G1) arrest was observed at 4h in OCI-AML3 and MV4-11 cell lines, indicating that cell cycle arrest occurred at an early time point which precedes the induction of cell death at 24h. The effects of CYC065 on proteins and gene expression were interrogated in the three cell lines. By using Western blotting, a downregulation of RNA polymerase II (RNAPII) was observed following treatment with CYC065 for 4h. Among the Bcell lymphoma 2 (BCL-2) family, a dramatic reduction of short half-life myeloid cell leukemia 1 (MCL-1) level was seen at both gene and protein level, highlighting a potential target inhibition of CYC065. A decrease in extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) was mutually observed in all cell lines, potentially reducing MCL-1 stability. The reduction of MCL-1 results in rapid induction of apoptosis. A decrease in gene expression of E2F transcription factor 1 (E2F1) was demonstrated, which potentially leads to G1 arrest. CYC065 in combination with other therapeutics; the selective BCL-2 inhibitor venetoclax, the conventional chemotherapy cytarabine, or the hypomethylating agent azacitidine was assessed for synergistic activity. Combination studies using CompuSyn software were performed to investigate the most efficacious drug combination ratios which were utilised for further synergistic investigations. Using various methods, the synergistic effect of CYC065/venetoclax, CYC065/cytarabine, and CYC065/azacitidine combinations were observed in all cell lines tested in this study. Rationally, the combination studies using these partners were continued in primary human AML samples. Primary human AML and normal haematopoietic cells were treated with two different concentrations of each therapeutic drug to encompass sensitive and resistant AML responses. The doses used were; 0.25µM or 0.5µM of CYC065, 0.025µM or 0.5µM of venetoclax, 0.01µM or 0.1µM of cytarabine, and 0.5µM or 2µM of azacitidine as a single agent or in combination for 72h. The data from six primary AML and three normal control samples are summarised aiming to observe a global effect of the treatments. Although not statistically significant, a decrease in cell number synchronously with an increase in annexin V-positive cells in the apoptosis assays, active caspase-3-positive cells in the active caspase-3 assays and an increase in the percentage of sub G0 population in cell cycle analysis was seen in the combination arms as compared with the single arms at both low and high concentrations in a dose-dependent manner. By contrast, normal cells experienced toxicity only at high concentrations of single and combination treatments with the exception of a single treatment of CYC065 and venetoclax. In the cell proliferation assays, at high concentrations, CYC065/azacitidine combination showed the synergistic effect in cell division arrest in primary AML but not in normal control samples, thus, a favourable therapeutic window was observed. Nevertheless, the concentrations of CYC065 and azacitidine needed to be tapered in order to reduce toxicity to normal cells. For the colony-forming unit (CFU) assays, although representing a small proportion of primary AML samples, a decrease in colony number, in which the majority of the colonies were CFU-monocyte (CFU-M), was seen in combination arms as compared with single treatments in a dose-dependent manner. The same effect was observed in normal samples in which a decline in colony number was seen regardless of colony type. High diversity of genetic mutations was observed for primary human AML samples. Considering the responses to the treatment of each sample as an individual, it was observed that the more complex the molecular genetic lesions or complexity of karyotype, the less efficacious the combination therapy responses observed. A variable response of these primary samples was the most plausible explanation for statistically insignificant results when a global effect of the treatments was evaluated. Nonetheless, an insufficient number of cases could introduce overinterpretation. Consequently, although the results were promising, it is inappropriate to draw any conclusions at present, with a larger cohort of patient samples required in order to fully evaluate the findings. In summary, our studies indicate the efficacy of the novel CDK2/9 inhibitor CYC065 as a single agent or in conjunction with frontline AML chemotherapeutics highlighting the potential of CYC065 for a novel therapeutic approach. Our work implicates the importance of individual genetic profiling as a critical step prior to initiating therapy for AML, highlighting the need for a more personalised medicine approach to improve outcomes in AML.