Proteasome Inhibitor Bortezomib Has Antitumour Activity Against Both CD34 Negative and CD34 Positive Acute Myeloid Leukemia Cells. (2006)

Abstract Proteasome inhibition represents a promising novel anticancer therapy, and Bortezomib is a highly selective reversible inhibitor of the 26S subunit of the proteasome complex. Acute Myeloid Leukemia (AML) is a heterogeneous group of diseases in which coexistence of leukemic cells at different stages of differentiation is frequently found, and CD34, a marker of immaturity, has been associated with drug resistance and poorer outcome. In order to explore the activity of Bortezomib in AML, we have evaluated its effectiveness against freshly isolated cells from 27 newly diagnosed AML patients (excluding APL). Mononuclear cells (MNC) were isolated from fresh bone marrow samples by Ficoll-Hipaque density sedimentation. The percentage of blasts after purification was 88±9% (Mean±SD). In order to assess the effective dose of Bortezomib in AML, four cell lines (HL-60, KG-1, HEL y MV4-11) were treated for 24h with increasing doses of Bortezomib. IC50 was 50nM (Range 10nM to 100nM). Based on these results, patients’ samples were treated with Bortezomib for 18 hours. Doxorubicin 1μM was also used for treatment in order to compare with Bortezomib activity in 23 paired samples. Using quadruple staining (Annexin V/CD33/CD34/CD45), the number of apoptotic cells was measured by multiparametric flow cytometry. At least 50.000 events were acquired in a FACScalibur cytometer (BDB). Annexin V+ (AV+) events were quantified over total cellularity, as well as in CD34+, CD34− blast cell fractions and normal residual lymphocytes. The percentage of apoptotic events was corrected according to the proportion of apoptotic cells in the control tube (absence of drug). The proportion of AV+ cells in the total blast cell population was 55±27%. CD34+ and CD34− blast cells populations were separately analyzed (only one leukemic cell population CD34+, or CD34− were detected in 7 and 13 cases, respectively; while in the remaining 6 cases both populations existed). Interestingly, Bortezomib showed similar activity on CD34+ (52±26%, n=13) and CD34− (55±26%, n=19) blast cell populations (p=0.68). Moreover, in the 6 cases in which CD34+ and CD34− leukemic cells coexisted, no difference was detected on the proportion of AV+ events after Bortezomib treatment in both cellular subsets (42±26% and 48±16% respectively, p=NS). In addition, the proportion of AV+ cells within the normal residual lymphocytes with bortezomid was significantly lower (20±12%) when compared with blast cells (p&lt; 0.01). Bortezomib was more effective for total blast cells compared to doxorubicin (56±32% vs 40±26%, respectively; p=0.009). Interestingly, when CD34+ and CD34− blast cell populations were separately analyzed, differences were only significant for the CD34+ populations (n=13) (52±26% vs 32±28% AV + cells for Bortezomib and doxorubicin, respectively; p=0.009), with no differences in either CD34− populations or normal residual lymphocytes. In summary, our results show a high antitumour activity of Bortezomib in AML blast cells, with similar efficacy against the more immature CD34+ and more mature CD34− leukemic cell subsets, while the toxicity against residual lymphocytes is significantly lower. These results support a therapeutic role for Bortezomib in adult Acute Myeloid Leukemia.