Chaetocin Exhibits Anti-Leukemia Activity Against Chronic Myeloid Leukemia Stem Cells

Abstract 1667

Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL kinase results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease, and implicating TKI-insensitive CML stem cells in relapse. Thus, there is great interest in identifying new therapeutic agents that can be combined with TKIs to eliminate TKI-insensitive CML stem cells that are not fully addicted to BCR-ABL. Chaetocin, a mycotoxin that inhibits histone methyltransferase SUV39H1, exhibits anticancer properties against myeloma and AML cells. To investigate a potential role for chaetocin in CML treatment, we first compared its cytotoxic effects with those of imatinib on TonB210, a murine hematopoietic cell line with doxycycline-inducible BCR-ABL expression that is dependent on BCR-ABL for survival and proliferation in the absence of exogenous IL-3. As expected, imatinib treatment produced cytotoxic effects in TonB210 cells induced to express BCR-ABL, but not in uninduced TonB210 cells, as assessed by trypan blue viability assays and Annexin V expression by flow cytometry. In contrast, chaetocin, displayed significant cytotoxicity against both TonB210 cells induced to express BCR-ABL and uninduced TonB210 cells. We then used a standard murine retroviral transduction system that models CML blast crisis (BC-CML) to study chaetocin effects on BCR-ABL expressing cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 48 hrs in chaetocin across a range of concentrations (0 – 400 nM). Chaetocin exhibited cytotoxic effects against GFP+/YFP+ LSKs as assessed by trypan blue, Annexin V, and colony forming assays. Chaetocin effects on CML stem cell self-renewal in vivo were assessed by performing secondary transplantation assays and limiting dilution analysis (LDA). GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to IC₅₀ concentrations of chaetocin and/or imatinib. Survival after transplantation and LSC frequency were compared in four treatment cohorts: Group 1 (untreated control), Group 2 (chaetocin), Group 3 (imatinib) and Group 4 (chaetocin + imatinib). Compared to untreated mice, survival was significantly prolonged in mice treated with imatinib alone and the survival benefit of imatinib was enhanced by co-treatment with chaetocin. LDA showed both chaetocin and imatinib treatment alone decreased LSC frequency (1 in 10,143 in chaetocin treated, and 1 in 95,782 in imatinib treated vs. 1 in 4,036 in untreated control) but that the combination of chaetocin and imatinib treatment resulted in a dramatic decrease in LSC frequency (1 in 293,628 in chaetocin + imatinib treatment vs. 1 in 4036 in untreated control). Chaetocin, imatinib, and chaetocin + imatinib treatment of normal murine hematopoietic stem cells resulted in only minimal cytotoxicity as assessed by trypan blue, Annexin V, and colony forming assays. Our findings suggest that chaetocin, or chaetocin-related compounds, might serve to complement TKI therapy in the treatment of CML.