Retention of Slow-Cycling CD34⁺ cells During Imatinib Treatment and Rapid Decline After 2nd ABL-TKI Treatment in Ph⁺ Leukemia Cells

Abstract 641

Several mathematical models and ex-vivo examinations suggested that imatinib (IM) therapy does not eradicate BCR-ABL-positive leukemia stem cells (LSCs). We investigated 47 CML-chronic phase cases using methods previously reported (Jamieson et al., N Engl J Med, 2004. and Abe et al., Int J Hematol, 2008) with FACSAria™ and quantitative RT-PCR of BCR-ABL among each sorted population; total mononuclear cells, HSC/Thy-1⁺, HSC/Thy-1^–, common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP). In optimal responders to IM therapy, BCR-ABL transcripts in the HSC populations (HSC/Thy-1⁺ and HSC/Thy-1^–) tended to be more retentive than other populations, while a gradual reduction was observed during the first 12 months in all populations. Moreover, the discrepancy of minimum residual diseases (MRD) between HSC and other populations became greater (about 2 log difference) in patients after longer IM therapy. We also prospectively investigated BCR-ABL transcripts in each population of 27 IM-resistant or -intolerant cases during treatment with the second-generation of ABL-tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib. Treatment with each inhibitor induced more rapid reduction of BCR-ABL transcripts even in the HSC population (CD34⁺CD38^–) during the first 6 months for IM-intolerant cases, and there was no statistical difference of MRD among each population in optimal responders to 2nd TKI therapy. In order to examine mechanisms of Ph⁺ LSC resistance to IM, Ph⁺ ALL patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγ^null (NOG) mice and cells derived from leukemic NOG mice were then co-cultured with S17-stromal cells using previously reported methods (Minami et al., PNAS, 2008). Despite complete dephosphorylations of BCR-ABL and its substrate CrkL, slow-cycling (Hoechst-33342^low/Pyronin-Y^low) CD34⁺ cells were insensitive to IM, which indicated that addiction of BCR-ABL activity is lower for survival in such quiescent cells. On the other hand, treatment with lower doses of 2nd TKIs induced not only growth inhibition but also substantial cell death including slow-cycling CD34⁺ population. More detailed biomarkers during pro-apoptotic status by 2nd TKI treatment are also under investigation. From comprehensive drug screening of small compounds in this co-culturing system, we also found several candidates involved with survival pathways in the cells. These results imply that 2nd TKI therapy can be a more promising approach than IM treatment for early reduction of Ph⁺ LSCs.