Page 185 - Haematologica March 2020

Targeting BCL2 and MAPK in AML
manner using CellTiter-Glo (CTG) assays after 72 h of drug treatment. The IC50 values of cobimetinib (range, 0.002 mM - 3.0 mM) did not correlate with either the status of RAS mutations or the basal levels of p-ERK1/2 deter- mined by flow cytometry (Table 1). To assess pharmaco- logical interactions between cobimetinib and venetoclax, incremental doses were applied based on the IC50 value of each drug. In seven of the 11 cell lines, combination of the agents elicited synergistic growth inhibition based on the Chou-Talalay method of analysis [combination index (CI) <0.8].25 Cell lines with IC50 values below the selected cut- off values (0.3 mM for cobimetinib20 and 0.1 mM for vene- toclax9) were defined as sensitive to the single agent. Patterns of response to single agents and the combination were distinct. Notably, while synergy was observed in both venetoclax-resistant (MOLM14, OCI-AML3, NB4) and cobimetinib-resistant cell lines (KG1, MOLM13), the lowest CI value (0.12) was seen in venetoclax- sensitive/cobimetinib-resistant AML cells (KG1) (Figure 1).
Cobimetinib and venetoclax demonstrate on-target suppression of cell proliferation and clonogenic potential of leukemia progenitors in a subset
of primary acute myeloid leukemia cells ex vivo
The anti-leukemia activities of cobimetinib and veneto- clax were examined in 18 primary samples with diverse genetic alterations, collected from patients with AML or spleen from PDX models (Table 2). Primary AML blasts were treated with cobimetinib and venetoclax alone or in combination at 0.1 mM for 5 days in LSC medium to main- tain the immature state of the leukemia cells.24 Cobimetinib alone induced minimal cell death (specific apoptosis, 6.7 ± 5.9%), which was significantly enhanced when the drug was given in combination with venetoclax (27.7 ± 20.2%, P=0.001) (Figure 2A, left). Cobimetinib inhibited cell proliferation in the majority of cases (34.2 ± 23.7%), and this suppression was more pronounced when the drug was combined with venetoclax (60.2 ± 28.8%, P<0.001) (Figure 2A, right). Venetoclax as a single agent reduced viable cell numbers by more than 50% in six cases (33.3%). Three of the four AML samples demon- strating over 50% growth inhibition by the cobimetinib
treatment carried the FLT3-ITD and/or D835 point muta- tion (AML 12, 13, and 17). As previously reported, IDH- mutant AML samples were highly sensitive to venetoclax as a single agent (AML 2 and 15). Over 60% (11 of 18) of the patients’ samples responded to the combination treat- ment, including those insensitive to either compound alone (AML 1, 8 and 11). Importantly, induction of apop- tosis in AML stem/progenitor CD34+CD38−CD123+ popu- lation following the combination treatment was observed in two out of four AML samples tested (Online Supplementary Figure S1). The clonogenic potential of myeloid progenitors was significantly suppressed by the combination (82.5 ± 20.0%), as compared to cobimetinib (38.3 ± 14.6%, P=0.01) or venetoclax (41.9 ± 18.6%, P<0.05) alone. Normal progenitor function was minimally affected (Figure 2B and Online Supplementary Figure S2).
To test the on-target efficacy of both agents, we devel- oped a 28-parameter mass cytometry [time-of-flight mass spectrometry (CyTOF)] panel comprising antibodies against surface antigens to define AML stem/progenitor fractions and intracellular proteins of the BCL2 family and various signaling pathways26 (Online Supplementary Table S1). The CyTOF study was performed in AML13 (sensi- tive to the combination) and AML14 (resistant to the com- bination) samples (Figure 2C). SPADE trees were built and annotated using all cell surface markers (Online Supplementary Table S1); the positive markers were includ- ed in the heat maps (Online Supplementary Figure S3). BCL2 protein levels were significantly enriched in CD34+ stem/progenitor cells compared to CD34– cells and BCL2 was expressed at a higher level in the venetoclax-sensitive sample (AML13) than in the venetoclax-resistant sample (AML14), consistent with our published data9 (Figure 2D). These results support the notion that venetoclax preferen- tially target LSC in AML. As previously reported, the can- cer signaling network relies on the manner in which can- cer cells respond to external stimuli rather than their basal phosphorylation state.27 Therefore, following cobimetinib treatment, we stimulated primary AML cells with granu- locyte colony-stimulating factor (G-CSF) or stem cell fac- tor (SCF) to study MEK downstream signaling pathways under conditions mimicking a cytokine-rich bone marrow
Table 1. Cytotoxicity of cobimetinib and venetoclax in acute myeloid leukemia cell lines.
Cell line
MOLM13
MOLM14 MV4;11 TF-1* OCI-AML3 OCI-AML2 THP1* KG1* NB4 U937* HL-60
IC (mM) 50
IC (mM) 50
Mutations
FLT3-ITD
FLT3-ITD
FLT3-ITD
NRAS, TP53
NPM1, DNMT3A, NRAS
DNMT3A 0.002
CI value
p-ERK (R-MFI)
Cobimetinib
Venetoclax
0.46
0.56 3.06 0.04 3.00 0.45
0.01
1.88 0.005 10.3 2.90 0.04 39.1 0.03 0.73 9.75 0.004
0.49 6.60
0.36 9.68 0.99 11.7 1.51 2.56 0.29 8.67 0.78 3.72 0.54 4.48 0.12 3.73 0.30 2.34 0.88 3.30 0.89 3.83
0.16 0.29 0.51 0.17
NRAS, TP53
ITGB8, SMC2 PML-RARA
PTPN11, WT1 CDKN2A, TP53, NRAS
Half maximal inhibitory concentration (IC50) values were calculated on the basis of the number of viable cells quantified by CTG assay. CI: combination index; R-MFI: relative median fluorescence intensity determined by the ratio of the signal in the antibody-stained cells/autofluorescence of unstained cells; ITD: internal tandem duplication. *Data on gene mutations are from Cancer Cell Line Encyclopedia: http://www.broadinstitute.org/ccle/home
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