BCL-2 Inhibition with ABT-199 Shows Efficacy in AML Cell Lines with Synergistic Effects upon Addition of the MEK Inhibitor GDC-0973 and Prolongs Survival in an NAS/BCL-2-Mediated Mouse Model of AML Post-MDS

Background and aims: BCL-2 activation plays a role in the progression of MDS to AML and BCL-2 inhibition may be a therapeutic target in such patients. ABT-199 is a small molecule which specifically inhibits BCL-2, approved for the treatment of CLL with TP53 mutation/17p deletion. Using the OCI-AML2 and MOLM13 AML cell lines, ABT-199 resistant cells have been derived to investigate the mechanism of drug resistance. The RAS/BCL-2 complex has been previously shown to co-localize in the mitochondria (Mito) in AML post-MDS mice and patients (Le Pogam Leuk Res 2013). To test the hypothesis that increased MCL-1 conferred ABT-199 resistance, OCI-MCL3 conditional lines in which MCL-1 could be switched on and off were studied for apoptosis and RAS/BCL-2 co-localization. In combination with our double transgenic mouse AML post-MDS preclinical model MRP8[NRASD12/BCL2] (Beurlet Blood 2013), we assessed the effect of ABT-199, and the MEK inhibitor GDC-0973.

Methods: The OCI-AML2 or MOLM13 ABT-199 resistant cell lines were transfected with either a control GFP (CON) or MCL-1-GFP tetracycline (OFF) inducible constructs and after treating overnight with doxycycline (DOX) (100 ng/ml) were incubated with a) no treatment, b) ABT-199 (1 µM) (Abbvie), c) GDC-0973 (1 µM) (Genentech) or d) ABT-199+GDC-0973 and a reagent for apoptosis, annexin V conjugated with a cyanine fluorescent red dye. Viability and cell death were followed using the Incucyte® Zoom System (Essen Bioscience). For RAS/BCL-2 co-localization studies, cells were labeled using antibodies specific for RAS (Alexa 647), BCL-2 (TRITC), Mito (Tom-20- Alexa 488) and plasma membrane (PM) (Ezrin-Alexa 488). In the MRP8[hNRASD12/hBCL2] model mice were treated at 3 weeks of age (13 vehicle, 18 ABT-199 (75mg/kg), 4 GDC-0973 (10mg/kg) and 6 ABT-199+GDC-0973) 3 times weekly by oral gavage for 33 days and followed for survival.

Results: Both OCI-AML2 ABT-199 sensitive and resistant cells had the RAS/BCL-2 complex localized primarily in the Mito. However, after ABT-199 treatment in the sensitive line the RAS/BCL-2 complex was still found in the Mito, whereas in the resistant line the complex was found in the PM. The OCI/GFP control (CON) cells showed resistance to ABT-199 with similar annexin V staining compared to untreated cells indicative of no change in apoptosis with similar viability in all conditions. The MCL-1 lines showed sensitivity to ABT-199 when MCL-1 was OFF (+DOX) and resistance when ON (-DOX) with an increased viability in the latter (Fig. 1A&B) and increased apoptosis in the former (Fig. 1C&D). The immunofluorescence profiles are summarized in Table 1, Fig. E&F. In the CON lines with no treatment, ABT-199 alone, ABT-199+GDC-0973 and the MCL-1 OFF cells (+ DOX), untreated and ABT-199 treated and the MCL-1 ON untreated cells the RAS/BCL-2 complex localized primarily in the Mito with a minority of PM localization. With GDC-0973 treatment of the CON and MCL-1 OFF lines or with the combined treatment of ABT-199+GDC-0973 of the MCL-1 OFF line and the MCL-1 ON treated with ABT-199 the complex was found equally in the Mito and PM. In the MCL-1 ON cells treated with GDC-0973 there was more complex in the PM than the Mito; with the combination of ABT-199+GDC-0973 no complex was detected, suggesting the clearance of the cells with the RAS/BCL-2 complex, which may explain the reduced viability and apoptosis profiles (Figure 1). The yellow staining cells in these panels were consistent with BCL-2 localizing in the PM and the Mito. The Mito localization of the RAS/BCL-2 complex indicates disease profile whereas PM localization resistance; regulation of MCL-1 appears to increase PM localization and increased resistance to ABT-199 or GDC-0973.

Survival of mice treated with ABT-199 was significantly longer (median 30 days) than those of untreated or vehicle (median 10 days) treated mice (p<0.008), underscoring drug efficacy. GDC-0973-treated mice died after 3-8 administrations at the same time as the vehicle-treated mice. Five of 6 mice treated with the combination of ABT-199+GDC0973 all survived until the end of treatment (15 administrations for 33 days), suggesting that the combination of ABT-199 and GDC-0973 extends lifespan (survival>75 days).

Conclusion:ABT-199 is effective in both in vitro AML lines and in vivo AML mice. The MEK inhibitor GDC-0973 synergises with ABT-199 in vitro and in vivo. RAS/BCL-2 co-localization may be a useful biomarker of response to treatments.

View largeDownload slide

View largeDownload slide

Close modal