An Autophagy Inhibitor, Chloroquine, Overcomes An Imatinib-Resistant T315I Mutant of Bcr/Abl, Whose Latent May Exist Before Imatinib Treatment.

Imatinib improved the prognosis of CML and Philadelphia chromosome-positive (Ph⁺) adult acute lymphocytic leukemia (ALL). Despite the stunning efficacy of this agent, a small number of patients develop resistance to imatinib. To overcome these resistant disease, second generation tyrosine kinase inhibitors (TKIs) such as dasatinib, nilotinib have been developed. The efficacy of these TKIs was shown against the various imatinib-resistant cells having gene mutations in Bcr/Abl, but could not inhibit against the gate keeper mutation, T315I. The T315I mutation is often observed in many case of aggressive phase of CML or Ph⁺ALL. Recently, some new drugs such as aurora kinase inhibitor and HDAC inhibitor, have been considered as the new drug against CML and Ph⁺ALL. It is known that HDAC inhibitor induced both apoptosis and autophagy in Bcr/Abl positive cells and that the block of autophagy accelerated apoptotic cell death. In recent studies, autophagy has been estimated to one of new target of treatment of Ph⁺ leukemia cells.

We established two Ph⁺ ALL cell lines, DPAL and DPAL/T315I, these cell lines from one clinical course of a patient have wild type and T315I mutation in Bcr/Abl, respectively. We analyzed the sequences of genes and the chromosomes of these cell lines. Using these cell lines as well as BaF/3 cell lines stably-transfected with wild type (BaF/3/wild) and mutated Bcr/Abl genes (BaF/3/T315I), we evaluated the effect of chloroquine diphosphate, one of the inhibitors of autophagy on cytotoxicity induced by imatinib or SAHA, one of a HDAC inhibitor.

This Ph⁺ ALL patient had double Ph⁺ chromosomes with additional chromosomal abnormalities at the first diagnosis. The patients received imatinib combined chemotherapy, then, relapsed after 3 months. T315I mutation was detected without additional chromosomal abnormality at relapse. DPAL was derived from bone marrow before imatinib treatment and DPAL/T315I was derived bone marrow at relapse after imatinib-combined chemotherapy. The DNA sequence analysis of DPAL/T315I cells revealed that one of two Philadelphia chromosomes had T315I mutation on Bcr/Abl. These result indicated that Bcr/Abl positive cells were heterogeneous clones before imatinib treatment in this clinical case. The most dominant populations was wild type Bcr/Abl clone with additional chromosomal abnormalities and one of minor population was T315I-positive cells without additional chromosomal abnormalities. This indicated the imatinib treatment eliminated the wild type Bcr/Abl-positive cells and subsequently T315I-positive cells increased. Other combination chemotherapies with stem cell transplantation could not overcome T315I-positive cells in this clinical course. So, to establish new treatment strategy, we studied about the effect of an autophagy inhibitor, chloroquine, against T315I-positive cells using our established cell lines. The combination treatment of imatinib with chloroquine increased the cell death of DPAL, BaF/3/wild, Ba/F3/T315I cells and inhibited the growth of DPAL/T315I cells. The combination treatment of a HDAC inhibitor, SAHA, with chloroquine induced cell death of not only DPAL cells but also DPAL/T315I cells. The phosphorylation of tyrosine on BCR/ABL did not change by chloroquine. The concentrations of intracellular ATP, assessed by luciferase assay, were decreased by chloroquine treatment.

From our established cell lines, it was suggested that Bcr/Abl-positive cells were heterogeneous clones in the Ph⁺ leukemia. Mono-treatment of TKIs against Bcr/Abl might permit the survival of tolerant clones such as T315I. The combination treatment of an autophagy inhibitor with TKIs or HDAC inhibitors were effective against Bcr/Abl-positive cells, even if T315I mutant cells. Our study indicated that autophagy is the protect system in Ph⁺ leukemia cells and block of autophagy might be one of new strategies for elimination of resistant clone of Bcr/Abl.

No relevant conflicts of interest to declare.