The BCR/ABL tyrosine kinase Imatinib mesylate (IM) is the mainstay of treatment for CML but does not eliminate primitive leukemia stem and progenitor cells. Residual leukemia cells in IM-treated patients are a potential source of relapse, and there is considerable interest in identifying additional therapeutic targets to selectively induce apoptosis in CML stem and progenitor cells. The Bcl-2 family of anti-apoptotic proteins control outer mitochondrial membrane integrity and play important roles in determining cellular susceptibility to apoptosis. Enhanced expression of Bcl-2 family genes may contribute to drug resistance in cancer cells. Several small molecule pan-Bcl-2 inhibitors are being evaluated for anti-cancer potential. However pan-Bcl2 inhibitors may be limited by toxicities to normal cells, and specific inhibition of individual Bcl-2 family members may be required for selective killing of leukemia cells. We performed a functional screen to evaluate the sensitivity of CML and normal CD34+ cells to inhibition of each of the 6 members of the Bcl-2 family. Expression levels of Bcl-2 family members in CML and normal CD34+ cells were measured by Q-PCR. We observed significant overexpression of Bcl2 (13.8-fold, p=0.0004), Mcl1 (3.5-fold, p=0.04), BclL1 (2.5-fold, p=0.028), and BclL2 (4.6-fold, p=0.01) in CML comparing with normal CD34+ cells. [CML (n=3) and CB (n=4)] CD34+ cells were separately transfected with a pool of 4 siRNA to each Bcl-2 family member (5μM, Smartpool siRNA, Dharmacon) and a pool of control siRNAs (ON-TARGETplus Non-Targeting Pool, Dharmacon). Transfection was performed by electroporation using the Amaxa Nucleofector system with the 96-well shuttle (Lonza). Replicate assays were conducted for each sample. Transfected cells were cultured in serum free medium with growth factors, with or without addition of IM (1μM). Q-PCR evaluation showed 50% to 80% inhibition of expression of targeted genes 24 hours after transfection. Cells were labeled with anti-CD34+ antibodies, Annexin V and DAPI. CD34+ cell apoptosis and viability were assessed 72 hours after transfection. CML CD34+ cells transfected with Mcl-1, Bcl2 and BclL1 siRNAs showed a significant increase in apoptotic cells (AnnexinV+ DAPI- cells). Only Mcl-1 siRNA resulted in significant reduction in the number of viable cells (DAPI- cells). Viability was further reduced in with addition of IM. In contrast knockdown of individual Bcl-2 family genes did not result in significant increase in apoptosis or loss of viability in CB CD34+ cells. To validate the anti-apoptotic role of Mcl-1 in CML progenitors, CML and CB CD34+ cells were transfected with a different pool of 4 Mcl-1 siRNAs (siGENOME Smartpool- Dharmacon, 5uM). Western blotting showed 80% reduction in Mcl-1 protein 48 hours after transfection compared with controls. We confirmed that Mcl-1 knockdown by siRNA resulted in increased apoptosis and reduced proliferation of CML but not CB progenitors (23±8% for CML vs 4.2±1.5% apoptosis for CB CD34+ cells). Induction of apoptosis by Mcl-1 inhibition was further enhanced in combination with IM treatment (48±15% for CML vs x7.2±3% apoptosis for CB progenitors). These results indicate that overexpression of Mcl-1 plays an important role in maintenance of CML progenitor cell survival, contributes to survival of CML progenitors treated with IM. Mcl-1 inhibition may offer a promising approach to selectively induce apoptosis in CML progenitors in combination with IM.
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