The Pan-Bcl-2 Family Inhibitor 97C1 Targets Blast Crisis Chronic Myeloid Leukemia Stem Cells but Spares Normal Cord Blood Progenitor Cells

Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML).

Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo.

As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo.

Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34⁺CD38⁺lin⁻ cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34⁺ cells derived from primary BC CML patient samples or primary CD34⁺ normal cord blood cells. In vitro drug responses were tested by culturing CD34⁺ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34⁺CD38⁺lin⁻ cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2^-/-y_c^-/- mice with CD34⁺ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS.

BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34⁺CD38⁺lin⁻ cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC₅₀ around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro.

Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34⁺CD38⁺lin⁻ cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34⁺CD38⁺lin⁻ cells in all hematopoietic organs analyzed.

Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells.

Jamieson:CoronadoBiosciences: Research Funding; CIRM: Research Funding.