ALBERT

Description: The BCR/ABL tyrosine kinase inhibitor imatinib mesylate (Gleevec, STI571; Novartis, Basel, Switzerland) has shown remarkable efficacy in the treatment of chronic myelogenous leukemia (CML), with a high proportion of patients achieving complete cytogenetic responses (CCRs). However, it is not clear whether remissions will be durable and whether imatinib mesylate can eliminate the malignant primitive progenitors in which the disease arises. We investigated whether residual BCR/ABL+ hematopoietic progenitors were present in patients who achieved CCRs with imatinib mesylate treatment. CD34+ progenitor cells were selected from bone marrow mononuclear cells (MNCs) and analyzed for the presence of the BCR/ABL fusion gene by fluorescence in situ hybridization (FISH). CD34+ cells were also plated in committed progenitor (colony-forming cell, or CFC) and primitive progenitor (long-term bone marrow culture-initiating cell, or LTCIC) cultures and resulting colonies analyzed for the presence of BCR/ABL+ cells by FISH. Using these assays, residual BCR/ABL+ progenitors were detected in all patients studied. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis demonstrated increased levels of BCR/ABL mRNA in CD34+ cells compared with total MNCs. Evaluation of samples collected at different time points demonstrated persistence of BCR/ABL+ progenitors despite continued treatment with imatinib mesylate. Our results indicate that inhibition of BCR/ABL tyrosine kinase activity by imatinib mesylate does not eliminate malignant primitive progenitors in CML patients. Patients in CCR with imatinib mesylate treatment need to be followed carefully to assess for risk of relapse.

Description: Imatinib mesylate (Gleevec) is a highly effective treatment for CML, but the long-term durability of response is unclear. We have identified residual BCR/ABL+ progenitors in patients achieving CCR on imatinib (Blood 2003, 101:4701). We have shown that imatinib inhibits CML progenitors through suppression of abnormally enhanced proliferation (Blood 2002, 99:3792) and that apoptosis is restricted to dividing cells with quiescent progenitors being resistant to apoptosis (Blood 2003, 102:652a). Here we have investigated whether growth factor (GF) stimulation of imatinib-treated CML progenitors could enhance proliferation and sensitivity to apoptosis and reduce the number of viable quiescent cells. CML and normal CD34+ cells were analyzed for apoptosis following culture in the presence or absence of imatinib (1μM) for 72 hours with either low (1μg/ml SCF and FL3, 0.2μg/ml IL6, G-CSF and IL3) or high (100μg/ml SCF and FL3, 20μg/ml IL6, G-CSF and IL3) GF concentrations. Cells were labeled with CFSE prior to culture to track cell division and allow separate examination of apoptosis in quiescent and proliferating cells. High GF conditions increased proliferation of both normal and CML CD34+ cells. Suppression of CML progenitor proliferation by imatinib was reduced in high GF conditions, compared to low GFs, when IL-3 was present (p=0.009), but not when IL3 was absent (p=0.065). Interestingly, normal CD34+ cells stimulated with high GFs demonstrated increased sensitivity to imatinib-mediated inhibition of proliferation, both with (p=0.005) and without IL3 (p=0.013). High GF conditions also resulted in reduced apoptosis of CML CD34+ cells, both with (p=0.003) and without IL-3 (p=0.001). In contrast, normal CD34+ cells demonstrated increased sensitivity to imatinib-induced apoptosis in high GFs. Imatinib did not induce apoptosis in undivided cells under either low or high GF conditions. For proliferating CML CD34+ cells we observed a reduction in apoptosis induced by imatinib in high GF conditions (+IL3: p=0.012; −IL3: p=0.019). The percent of cells used to initiate culture that remained as viable, undivided cells following exposure to imatinib was calculated. There was a trend towards a reduction in the number of quiescent, viable CML CD34+ cells after imatinib treatment in high GF conditions (p=0.071). However, high GF conditions also led to a reduction in the number of quiescent, viable normal cells. In conclusion, we show that high GF conditions counter imatinib-mediated proliferation suppression in CML CD34+ cells but also protect cells from apoptosis. GF stimulation also enhances the sensitivity of normal CD34+ cells to imatinib. A trend towards a reduction in viable, quiescent CML progenitors was seen, even with short-term GF exposure, supporting further evaluation of the use of GF stimulation to reduce residual CML progenitors during imatinib treatment. Effect of Imatinib on CD34+ Cells Low Growth Factors High Growth Factors ¹relative to cells not exposed to imatinib. ²CFSE compared to starting population: less=divided; same=undivided. ³% of input cells that remain undivided & non-apoptotic with imatinib. +IL3 −IL3 +IL3 −IL3 Proliferation Suppression¹ CML 54 ± 2 56 ± 2 45 ± 2 52 ± 1 Normal 25 ± 4 30 ± 4 51 ± 1 53 ± 2 Apoptosis Increase¹ CML 23 ± 2 29 ± 3 17 ± 3 20 ± 3 Normal 3.4 ± 3.6 4.4 ± 3.9 13 ± 4 13 ± 2 CML-Divided² 27 ± 3 41 ± 8 9.9 ± 1.8 8.6 ± 2.5 CML-Undivided² −4.1 ± 3.7 −2.8 ± 4.1 −1.4 ± 5.5 0.6 ± 5.0 % remaining undivided and viable³ CML 25 ± 7 22 ± 5 20 ± 8 22 ± 8 Normal 46 ± 11 46 ± 12 25 ± 2 37 ± 7

Description: Chronic myelogenous leukemia (CML) results from malignant transformation of a primitive hematopoietic cell by the BCR/ABL oncogene. The breakpoint cluster region/ABL (BCR/ABL) tyrosine kinase inhibitor imatinib mesylate (imatinib) is highly effective in inducing remissions in CML. However, the effects of imatinib on intracellular signaling in primary progenitor cells are not well described. We show that imatinib exposure resulted in a significant dose-responsive reduction in BCR/ABL kinase activity in CML CD34+ cells. However, imatinib treatment resulted in an increase in activity of p42/44 mitogen-activated protein kinase (MAPK), an important downstream effector of BCR/ABL. Increased MAPK activity was growth factor dependent. Pharmacologic inhibition of MAPK using MAPK/extracellular signal–regulated kinase kinase–1/2 (MEK-1/2) inhibitors significantly reduced CML progenitor proliferation. Combined treatment with a MEK-1/2 inhibitor and imatinib significantly increased suppression of CML progenitors compared with either inhibitor alone. In contrast, imatinib treatment resulted in a small reduction in AKT activity. Combined treatment with a phosphatidylinositol-3 (PI-3) kinase inhibitor and imatinib significantly increased suppression of CML progenitor growth compared with either inhibitor alone. We conclude that inhibition of BCR/ABL kinase activity in CML progenitors by imatinib results in a growth factor-dependent compensatory increase in MAPK activity and in only partial inhibition of PI-3 kinase activity. These mechanisms may contribute to incomplete elimination of CML progenitors by imatinib. (Blood. 2004;103:3167-3174)

Description: The therapeutic success of imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by persistence of malignant stem cells. Mechanisms contributing to incomplete elimination of CML stem cells include resistance to proapoptotic effects of IM, persistent signaling through growth stimulating pathways and Abl kinase mutations resulting in IM resistance. We investigated whether nilotinib, a more potent Bcr-Abl tyrosine kinase (TK) inhibitor than IM, could more effectively target CML progenitors. CD34+ progenitors from CML patients and healthy donors were cultured with nilotinib (0–10μM) and IM (0–5μM) in growth factor (GF) containing medium. After 96h incubation, cells were harvested and assayed for colony-forming-(CFC) and long-term culture initiating cells (LTCIC). CD34+38- primitive progenitor cells (PPC) and CD34+38+ committed progenitor cells (CPC) labeled with CFSE were incubated under similar conditions and evaluated for proliferation and apoptosis assays by annexin-V staining and FACS analysis. The IC50 for CML LTCIC-suppression was 0.9μM for nilotinib and 0.8μM for IM. Normal LTCIC were not significantly suppressed at doses below 10μM nilotinib and 5μM IM. The IC50 for CML CFC suppression for nilotinib was 6.4μM and 1.0μM for IM. Normal CFC showed less growth inhibition and the IC50 was not reached at the dose range tested. CFSE assays indicated dose-dependent antiproliferative activity of both compounds with IC50-values of 3.0μM nilotinib and 1.8μM IM for CML PPC, and 8.2μM nilotinib and 4.0μM IM for CML CPC. In CML PPC, apoptosis significantly increased following nilotinib treatment (8±2.1% [control] to 22±2.7% [10μM], n=4, p=.015). IM treatment also increased apoptosis in CML PPC (to 35±3.8% [5μM], n=4, p=.0008). In CML CPC, apoptosis increased from 33±2.3% [control] to 50±1.7% (n=4, p=.002) with 10μM nilotinib, and to 53±2.1% (n=4, p=.001) with 5μM IM exposure. The effects on IM and nilotinib on Bcr-Abl-TK activity were investigated by Western blotting with anti-CrkL antibodies after overnight drug exposure of CML CD34+ cells in GF containing media. Importantly, 0.25μM nilotinib significantly reduced P-CrkL levels (from 85.7±4.1% [control] to 13.9±4.5%, n=4, p=.000002), whereas a higher concentration of IM (1–5μM) was needed to achieve similar inhibition. Nilotinib resulted in increased MAPK activity (to 13.3-fold [5μM]) in CML CD34+ cells. MAPK kinase activity was also increased following treatment with IM (to 22.7-fold [5μM]). P-AKT and P-STAT5 levels were not significantly changed in response to either drug. In contrast incubation with either drug in the absence of GF resulted in inhibition of MAPK, Akt and STAT5 activity in CML CD34+ cells. In conclusion, nilotinib is significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors but does not induce greater suppression of progenitor growth. As with IM, inhibition of cell division represents the predominant mode of inhibition of progenitor growth. Nilotinib or IM treatment inhibits Bcr-Abl dependent MAPK, STAT5 and Akt activity, but does not inhibit these signaling mechanisms in CML progenitors in the presence of GF. Our results suggest that combined inhibition of Bcr-Abl TK and additional signaling mechanisms may be required to achieve elimination of CML progenitors by targeted therapies.

Description: Imatinib mesylate (STI571) is a promising new treatment for chronic myelogenous leukemia (CML). The effect of imatinib mesylate on primitive malignant progenitors in CML has not been evaluated, and it is not clear whether suppression of progenitor growth represents inhibition of increased proliferation, induction of apoptosis, or both. We demonstrated here that in vitro exposure to concentrations of imatinib mesylate usually achieved in patients (1-2 μM) for 96 hours inhibited BCR/ABL-positive primitive progenitors (6-week long-term culture–initiating cells [LTCICs]) as well as committed progenitors (colony-forming cells [CFCs]). No suppression of normal LTCICs and significantly less suppression of normal CFCs were observed. A higher concentration of imatinib mesylate (5 μM) did not significantly increase suppression of CML or normal LTCICs but did increase suppression of CML CFCs, and to a lesser extent, normal CFCs. Analysis of cell division using the fluorescent dye carboxyfluorescein diacetate succinimidyl ester indicated that imatinib mesylate (1-2 μM) inhibits cycling of CML primitive (CD34+CD38−) and committed (CD34+CD38+) progenitors to a much greater extent than normal cells. Conversely, treatment with 1 to 2 μM imatinib mesylate did not significantly increase the percentage of cells undergoing apoptosis. Although a higher concentration of imatinib mesylate (5 μM) led to an increase in apoptosis of CML cells, apoptosis also increased in normal samples. In summary, at clinically relevant concentrations, imatinib mesylate selectively suppresses CML primitive progenitors by reversing abnormally increased proliferation but does not significantly increase apoptosis. These results suggest that inhibition of Bcr-Abl tyrosine kinase by imatinib mesylate restores normal hematopoiesis by removing the proliferative advantage of CML progenitors but that elimination of all CML progenitors may not occur.

Description: While imatinib mesylate is a highly effective treatment for CML, there is accumulating evidence that it may not adequately target quiescent malignant HSCs. In vitro exposure to imatinib inhibits CML progenitor growth primarily through suppression of abnormally enhanced proliferation. Apoptosis appears to be restricted to dividing cells while quiescent progenitors are resistant to apoptosis. One approach to more effectively enhance the sensitivity of HSCs to imatinib may be to induce them to cycle using hematopoietic growth factors (GF). We have shown that exposure of CML CD34+ progenitors to imatinib (1μM) in high GF conditions (100ng/ml SCF and FL3, 20ng/ml IL6, G-CSF and IL3) reduced the total number of viable, undivided cells compared to control cells cultured in 100-fold lower GF conditions (low GF). High GF treated cells were more proliferative but less sensitive to imatinib-mediated apoptosis (Blood2004, 104:2967). We hypothesized that pre-stimulation with high GF prior to imatinib exposure would further reduce viable, non-dividing CML progenitors. CML CD34+ cells were cultured in high GF for 48 hours and then exposed to imatinib (1μM) for 48 hours in either high or low GF conditions. Compared to cells exposed to imatinib without any pre-stimulation, high GF pre-stimulation significantly reduced imatinib-mediated inhibition of proliferation in both low GF (22±5%, p=0.0009) and high GF (18±3%; p=0.0003). Pre-stimulation decreased imatinib-mediated apoptosis when compared to the same conditions with no pre-stimulation [19±2% for imatinib treatment in low GF (p