Overcoming BCR::ABL1 dependent and independent survival mechanisms in chronic myeloid leukaemia using a multi-kinase targeting approach

BackgroundDespite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture.MethodsCML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR.ResultsBy targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred.ConclusionBMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence. Blood is made in the spongy inner most section of the bone, called the bone marrow. The bone marrow is where normal blood stem cells live that are responsible for producing the different blood cell types; white blood cells (fight infections), red blood cells (carrying oxygen around the body), platelets (blood clotting) and other cells which support this process. Chronic myeloid leukaemia (CML) is a type of blood cancer that starts in the bone marrow. CML occurs when a normal blood stem cell becomes damaged, forming a leukaemia stem cell (LSC), leading to blood cancer. LSCs multiply and generate many faulty cancerous white blood cells that do not work properly. Patients are treated with a drug called imatinib, which reduces the number of cancerous cells circulating in the body. In many cases, this treatment is not enough to cure the disease because the bone marrow protects the LSCs from the drug meaning patients must remain on long term treatment. This work has discovered one of the ways in which the bone marrow protects LSCs from treatments and has used this knowledge to test new drugs that stop this protection. Our findings show that by combining two drugs, one that overcomes this protection and one that directly targets the cancerous cells, we can destroy more of the LSCs. These findings are a step closer towards a cure for CML and could improve treatment for patients in the future.8kTkWYvNitSmKGMPj2MgjoVideo Abstract