Page 184 - Haematologica March 2020

L. Han et al.
leukemogenesis may deliver the greatest clinical efficacy. The anti-apoptotic protein BCL2 has been studied exten- sively for its role in leukemic transformation and chemore- sistance. BCL2 is highly expressed in AML leukemia stem cells (LSC) containing low levels of reactive oxygen species which are resistant to chemotherapy.4,5 BCL2 inhibitors have been shown to eradicate AML LSC and sensitize chronic myeloid leukemia LSC to tyrosine kinase inhibitors. Oncogenic dependency on BCL2 was found in AML patients carrying mutations in IDH1 and IDH2.6,7 A recent study used BH3 profiling to discover that co-inhibition of BCL2 with tyrosine kinase inhibitors facilitated eradication of genetically diverse AML in patient-derived xenograft (PDX) models.8 We have reported the anti-leukemia potency of venetoclax (ABT-199/GDC-0199), an orally bioavailable BH3 mimetic that selectively binds with high affinity to BCL2, but lacks affinity for BCL-XL and MCL1, in AML models.9 In a phase II clinical trial, venetoclax monotherapy had clinical activity in patients with relapsed or refractory AML with a tolerable safety profile.10 However, the inability of venetoclax to inhibit MCL1 causes resistance in leukemia
cells that require MCL1 for survival.11,12
The RAF/MEK/ERK (MAPK) cascade, a major effector
pathway activated in 70%-80% of patients with AML, is activated by upstream mutant proteins such as FLT3, KIT, and RAS.13-15 The MAPK pathway regulates BCL2 family proteins by stabilizing anti-apoptotic MCL111,16 and inacti- vating pro-apoptotic BIM (BCL2L11).17 Monotherapy with MEK inhibitors has had limited clinical efficacy.15 Recently it was shown that MAPK signaling activation contributed to primary resistance to an IDH2 inhibitor18 and acquired resist- ance to venetoclax,19 suggesting that combination regimens that include MEK inhibitors could be efficacious in these patients. Cobimetinib (GDC-0973) is an allosteric MEK inhibitor with antitumor activity in BRAF- and KRAS- mutant tumor cells,20 and was recently approved to treat patients with metastatic melanoma. Its anti-AML efficacy, particularly in combination with venetoclax, is unknown.
Furthermore, biomarkers predictive of response to this combination in AML are unknown. Suppression of down- stream pERK does not predict sensitivity to MEK inhibi- tion.21 In melanoma, it has been demonstrated that mTORC1/2 and pS6 activities are associated with acquired resistance to MEK inhibitors and suppression of pS6 may serve as a biomarker to predict clinical response to MEK inhibitors.22,23 The role of pS6 in response to MEK and BCL2 inhibition has not been addressed.
In this study, we evaluated the anti-leukemia effects of concomitant BCL2 and MAPK blockade by venetoclax and cobimetinib in AML cell lines, primary patients’ samples, and xenograft murine models. Through the use of reverse-phase protein arrays (RPPA) and RNA sequencing, we identified pharmacodynamic markers that correlated with the efficacy of the combination treatment, in particular activated pS6 (Ser235/236), which discriminated combination-sensitive from -insensitive AML cells. Our data support the rationale for dual inhibition of the BCL2 and MEK pathways.
Methods
Patients’ samples, acute myeloid leukemia cell lines, and reagents
Bone marrow and peripheral blood samples were collected from patients with AML or healthy donors after informed con-
sent had been obtained in accordance with the Institutional Review Board of The University of Texas MD Anderson Cancer Center. The cell line culture methodology is described in the Online Supplementary Methods.
Assays and other methods
Details of the CellTiter-Glo proliferation assay, colony-form- ing cell assay, electrochemiluminescent enzyme-linked immunosorbent assay, RPPA, and RNA sequencing are provided in the Online Supplementary Methods. Antibody conjugation for mass cytometry staining and the spanning-tree progression analysis of density-normalized events (SPADE) analysis are also explained in detail in the Online Supplementary Methods.
Apoptosis in primary acute myeloid leukemia samples
As previously reported,24 18 primary AML peripheral blood mononuclear cells or AML PDX samples were cultured in LSC medium. Viable AML CD45dim blast cells were enumerated by using CountBright counting beads (Cat. C36950; Invitrogen, Carlsbad, CA, USA) with concurrent annexin V and DAPI detection on a Gallios Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA). Data analysis and additional details are included in the Online Supplementary Methods.
In vivo study of cobimetinib and venetoclax in acute myeloid leukemia xenograft mouse models
The animal studies were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee at MD Anderson. Nonobese diabetic/severe com- bined immunodeficient gamma IL3-GM-SF (NSG-SGM3 or NSGS) mice (female, 8-10 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The mice were injected intravenously with OCI-AML3-Luci-GFP (1.0×106) cells, which were lentivirally transduced with firefly luciferase. Leukemia engraftment was confirmed 1 week after injection through a noninvasive in vivo bioluminescence imaging (BLI) system (Xenogen, Alameda, CA, USA) after injection of a D- luciferin (4 mg/mouse) substrate. Mice were distributed into four groups (11 mice/group) with comparable tumor burden and dosed daily for 4 weeks with one of the following oral preparations: vehicle, cobimetinib (10 mg/kg), venetoclax (100 mg/kg), or cobimetinib plus venetoclax. BLI was performed weekly to determine the extent of engraftment. Survival was monitored as an endpoint. A similar MOLM13 model is described in the Online Supplementary Methods.
Statistical analyses
The Student t-test was used to analyze the statistical signifi- cance of differences between groups, both in vitro and in vivo. All statistical tests were two-sided, and the results are expressed as the mean ± standard deviation. A P value ≤0.05 was considered statistically significant. The RPPA and RNA-sequencing data analysis are described in the Online Supplementary Methods.
Results
Cobimetinib and venetoclax demonstrate synergistic anti-leukemia efficacy in acute myeloid leukemia cell lines in vitro
To assess the anti-leukemia activity of cobimetinib and venetoclax as single agents or in combination, we studied their effects on cell proliferation of 11 AML cell lines (Table 1). The median inhibitory concentration (IC50) val- ues of single agents were determined in a dose-response
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