Developing a new therapeutic for childhood acute myeloid leukaemia
Chief Investigator: Dr Mingfeng Yu
Funding Amount: $ 35,000
Recipient: University of South Australia
In Australia, acute myeloid leukaemia (AML) is the second leading cause of cancer death in children. Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are the most frequent genetic alterations in AML, and are associated with poor prognosis. Thus, FLT3 has emerged as a therapeutic target for treating AML. We have recently identified a highly potent FLT3 inhibitor that displays a selectivity for this kinase superior to known FLT3 inhibitors under development. This project will further evaluate the anti-cancer efficacy of this inhibitor in animal models of AML, and a successful outcome will support its advancement for treating childhood AML.
Researchers: Mingfeng Yu, Shudong Wang, Jasmine Karanjia
Research Completed: 2020
Acute myeloid leukaemia (AML) is a type of blood cancer that accounts for approximately 10% of deaths among Australian children diagnosed with cancer and constitutes the second leading cause of their cancer deaths. With current chemotherapeutic treatments, cure rates for the children with relapsed or refractory AML remain unacceptably low—in a range of 17-33%. A recent comprehensive characterisation of paediatric AML at the molecular level has identified the FLT3 gene as being most frequently mutated. Among the known mutations, FLT3-ITD mutation has lately been defined as a driver lesion in human AML and validated as a therapeutic target for its treatment. Our project was aimed at advancing CDDD5-5, a highly selective and potent inhibitor of FLT3, towards preclinical development.
As a first step, we successfully scaled up the synthesis of CDDD5-5 by optimisation of its synthetic route and several workup procedures, affording more than 20 grams with an analytical purity of ≥ 99%. With sufficient amount of CDDD5-5, we first assessed its selectivity for FLT3 over a panel of 369 human kinase at a concentration of 1 µM, and found FLT3 is the one and only kinase whose kinase activity was inhibited by > 90%. To the best of our knowledge, CDDD5-5 is the most selective FLT3 inhibitor reported so far. Furthermore, we tested CDDD5-5 against a panel of eight FLT3 mutants, and found that it even more potently suppressed all of them than wide-type FLT3. In parallel, we evaluated the anti-proliferative effect of CDDD5-5 in a wide selection of 39 cancer cell lines, ranging from leukemias to solid tumours originating from ovary, lung, prostate and breast. We found that the cell lines harbouring FLT3-ITD mutation are most sensitive to the compound. One of such examples is MV4-11 AML cell line. Hence, this cell line was selected to study the anti-proliferative effects of CDDD5-5 in vitro and in vivo.
To investigate the mechanism of anti-proliferative action of CDDD5-5, we used a series of cell-based assays, including apoptosis detection, cell cycle analysis and western blotting. In comparison with the control experiments where CDDD5-5 was not included, we found that the addition of the compound (1) induced cell death, (2) arrested cells at G1 phase of the cell cycle, and (3) dampened the signal transduction downstream of FLT3. These effects became profounder as the compound concentration increased. Taken together, these findings support an FLT3-targeted mechanism of anti-proliferative action of CDDD5-5 in MV4-11 cells.
At the core of our project was the assessment of anti-cancer efficacy of CDDD5-5 in an AML xenograft mouse model that had been previously established with our group. Prior to this, we had profiled the biopharmaceutical and pharmacokinetic properties of CDDD5-5, and confirmed that the compound possesses a suitable combination of these properties for its progression to animal models of cancer. With these preliminary data in hand, we first performed a study using BALB/c nude mice to determine the highest dose of CDDD5-5 that can be administered without causing significant signs of toxicity. This dose is defined as a maximum tolerated dose (MTD). This MTD (200 mg/kg) was orally administered once a day to mice which had been inoculated subcutaneously with MV4-11 cells into their hind flanks and developed tumours of appropriate sizes. After 28-day dosing, we found the tumours regressed significantly and no mice died or showed any overt clinical signs of toxicity at the conclusion of the anti-cancer efficacy study (i.e., 70 days). These results suggest CDDD5-5 holds great promise for treating AML, and warrant further preclinical assessments which are beyond the scope of our project. Overall, we have achieved all of the three objectives set out in our project, including scale-up preparation of CDDD5-5, confirmation of the FLT3-targeted mechanism of its anti-proliferative effect in MV4-11 cells, and determination of its anti-cancer efficacy in the MV4-11-xenografted mouse model.
- CDDD5-5 is a highly selective and potent inhibitor of FLT3, and exhibits inhibitory potency against the cell lines harbouring FLT3-ITD mutation.
- In MV4-11 cells, CDDD5-5 induces apoptosis, halts the cell cycle in G1 phase, and exerts its anti-proliferative effect via targeting FLT3.
- In an MV4-11-xenografted mouse model, CDDD5-5 demonstrates its potent anti-cancer efficacy without causing any overt clinical signs of toxicity.
 Long, Y, Yu, M, Ochnik, AM, Karanjia, JD, Basnet, SKC, Kebede, AA, Kou, L & Wang, S, ‘Discovery of novel 4-azaaryl-N-phenylpyrimidin-2-amine derivatives as potent and selective FLT3 inhibitors for acute myeloid leukaemia with FLT3 mutations’, manuscript in preparation.
 Karanjia, JD, Ochnik, AM, Long, Y, Yu, M, Teo, T, Albrecht, H, Milne, R & Wang, S, ‘Advances in the evolution of FLT3 inhibitors for acute myeloid leukemia therapy’, manuscript in preparation.