Hsp-90 Inhibition Decreases Survival Of Bcr-Abl T315i Leukemic Stem Cells In Mice.

Although advances have been made in the development of novel molecularly targeted drugs, a major therapeutic challenge in the treatment of patients with Philadelphia chromosome positive (Ph+) leukemia includes understanding how to target the leukemic stem cell. We used the bone marrow transplant (BMT) model of chronic myelogenous leukemia (CML) to study effects of imatinib mesylate and the novel, orally active heat shock protein 90 (Hsp90) inhibitor, IPI-504, on leukemic stem cells, based on our observation that unlike imatinib, IPI-504, prolongs survival in a murine model of drug-resistant T315I BCR-ABL-induced CML. We first identified BCR-ABL-expressing hematopoietic stem cells (HSCs) (Lin-c-Kit+Sca-1+) in mouse bone marrow as CML stem cells, as these cells sorted out by FACS from primary CML mice are sufficient to confer leukemia in recipient mice. We then investigated the effects of imatinib and IPI-504 on survival of leukemic stem cells from BCR-ABL T315I induced CML. Bone marrow cells from mice with T315I-induced CML were cultured under conditions that support survival and growth of stem cells, with or without IPI-504 or imatinib. FACS analysis of GFP+Lin-c-Kit+Sca-1+ cells showed that imatinib treatment did not lower the percentage and the number of leukemia stem cells, whereas IPI-504 treatment had a dramatic inhibitory effect on this population (p<0.001) at therapeutically achievable doses. To determine whether IPI-504 attenuates development of leukemia by specifically inhibiting stem cell survival, GFP+Lin-c-Kit+Sca-1+ cells were sorted from bone marrow of mice with BCR-ABL T315I-induced CML, and cultured with a placebo or IPI-504. When these cells were transferred into lethally-irradiated recipient mice, FACS analysis showed that myeloid leukemia cells were present in mice receiving the placebo-treated leukemic stem cells but not in mice receiving the IPI-504 treated leukemic stem cells. To examine whether IPI-504 inhibits leukemia stem cells in vivo, mice with BCR-ABL-T315I-induced CML were treated with a placebo, imatinib, or orally administered IPI-504 for six days. Bone marrow cells were analyzed by FACS for GFP+Lin-c-Kit+Sca-1+ cells. Consistent with the in vitro results, imatinib treatment did not lower the percentage and number of leukemia stem cells, as compared with the untreated group, whereas IPI-504 treatment had a dramatic inhibitory effect on the stem cells. Analysis of bone marrow from non-leukemic mice treated with IPI-504 for two weeks showed no change in levels of Lin-c-Kit+Sca-1+ cells, indicating that IPI-504 treatment did not inhibit survival of normal HSCs. These results provide a rationale for use of an Hsp90 inhibitor as a first-line treatment to inhibit leukemia stem cells and prevent emergence of imatininb-resistant clones in patients.