Page 75 - 2022_01-Haematologica-web

Targeting AML MCL-1 re-sensitizes BCL-2 inhibition
venetoclax-resistant OCI-AML3 (Figure 3C), MCL-1-OE Molm13 (Figure 3D), and MV4-11 cells (Online Supplementary Figure S2C). This was also the case in MV4-11R cells (Figure 3E).
We then tested CDK9 inhibitor AZD4573. AZD4573 decreased MCL-1 expression and synergized with veneto- clax targeting OCI-AML3 cells (Online Supplementary Figure S2D). This combination was also highly synergistic against MV4-11 and MV4-11R cells (Online Supplementary Figure S2E). In order to demonstrate that MCL-1 is indeed the critical target regulated by CDK9, we treated MCL-1- OE Molm13 and MV4-11 cells with AZD4573 and found that, similar to the MCL-1 inhibitor AZD5991, MCL-1 OE cells were more resistant than control cells to AZD4573. Furthermore, the combination of venetoclax and AZD4573 was highly synergistic in MCL-1-OE Molm13 and MV4-11 cells (Online Supplementary Figure S2F).
We next treated OCI-AML3 cells with venetoclax, AZD5991, or the combination and determined interac- tions between anti-apoptotic BCL-2, MCL-1, and BCL-XL with the pro-apoptotic activator BIM and effectors BAX and BAK by co-immunoprecipitation to potentially under- stand the mechanisms of synergy. We observed that BCL- 2 was bound to BIM and BAX (Figure 4A), MCL-1 was bound to BIM and BAK (Figure 4B), and BCL-XL was bound to all three (Figure 4C). Venetoclax treatment decreased BCL-2/BIM and BCL2/BAX interactions, but increased MCL-1/BIM, BCL-XL/BAX, and BCL-XL/BAK interactions. AZD5991 treatment decreased MCL-1/BIM interaction, but increased BCL-2/BIM and MCL-1/BAK interactions. When the two drugs were combined, BCL- 2/BIM and BCL2/BAX interactions were largely dimin- ished, MCL-1/BIM interaction decreased, and the single agent treatment-mediated increases of BCL-XL/BAX, BCL-XL/BAK, and MCL-1/BAK interactions were abrogat- ed (Figure 4A to C), suggesting that the combinatorial cooperative release of activator and effector BCL-2 family proteins likely contributed to the synergy in apoptosis induction (Figure 4D).
In order to determine the contribution of metabolism and leukemia-stroma interactions on the observed syner- gism, we treated OCI-AML3 cells with venetoclax in the presence of the OxPhos inhibitor IACS-1075942 or the CXCR4 inhibitor BL-8040 (4F-benzoyl-TN14003).43 As expected, MSC co-culture protected AML cells from vene- toclax-, AZD5991-, or IACS-10759-induced apoptosis (Figure 4E). While suppressing OCI-AML3 migration and adhesion to MSC, BL-8040 did not affect cell viability (Figure 4E and F). Under MSC co-culture, venetoclax activ- ity was minimally enhanced by OxPhos or CXCR4 inhibi- tion, but markedly augmented by combinatorial OxPhos and CXCR4 inhibition. Maximal apoptosis induction of OCI-AML3 was observed by combined MCL-1 and BCL- 2 inhibition (Figure 4E). These results support that cooper- ative release of pro- from anti-apoptotic BCL-2 family pro- teins and inhibition of cell metabolism and key stromal microenvironmental mechanisms all contributed to the synergism of co-targeting MCL-1 and BCL-2 in AML cells.
Combined BCL-2 and MCL-1 inhibition synergistically induces apoptosis in primary acute myeloid leukemia (AML) cells and AML stem/progenitor cells
Primary AML cells were cultured with BM-derived MSC and treated with venetoclax, AZD5991, AZD4573, venetoclax plus AZD5991, or venetoclax plus AZD4573.
Apoptosis and viable cell counts of AML blast cells and CD34+ stem/progenitor cells were assessed and expressed as EC50 (Figure 5A) or IC50 (Online Supplementary Figure S3) for each agent used alone or in combination, as indicated. The patient characteristics, including mutations, veneto- clax treatment status, and cytogenetics/risk categories, are shown in Figure 5B and Online Supplementary Table S1. Responses to venetoclax, AZD5991, or AZD4573 varied across the patient samples. Compared with the single-agent treatments, the venetoclax plus AZD5991 and venetoclax plus AZD4573 combinations were markedly more effective in inducing apoptosis and decreasing viable cell numbers (markedly lower EC50 and IC50 values, respectively) in not only blasts, but also CD34+, CD34+CD38+/CD34+CD38- and CD34+CD38+CD123+/CD34+CD38-CD123+ stem/progeni- tor cells in all primary AML samples, including those from venetoclax-resistant/relapsed patients (Figure 5A and B; Online Supplementary Figure S3; Online Supplementary Table S1) regardless of mutational status and cytogenetics.
Among samples 7, 8, 11, and 12 from venetoclax or venetoclax/decitabine resistant/relapsed patients, three (7, 8, and 12) had relatively high venetoclax EC50 values. Samples 3 and 10 that exhibited relatively high venetoclax EC50 values were from resistant/relapsed patients who received venetoclax after sampling and were resistant or relapsed (Figure 5A and B; Online Supplementary Table S1), suggesting that our in vitro system mirrored clinical responses. Patient samples with high venetoclax EC50 val- ues tended to have high AZD5991 EC50 values, suggesting that AZD5991 monotherapy would be less effective in venetoclax-resistant patients. However, more samples are needed to confirm our conclusion. We observed that sam- ples with WT1 (3 and 12) or BCORL1 (4 and 7) mutations were the most resistant to AZD5991 and AZD4573, respectively (Figure 5A and B). Experiments with primary AML cells treated without MSC co-culture yielded similar results (Online Supplementary Figure S4), but these cells were generally more sensitive to the treatments support- ing the protective role of MSC. The CI values for the com- binations for each AML cell population are shown in the Online Supplementary Table S3.
Co-targeting BCL-2 and MCL-1 exerts pronounced anti-leukemia activity in a patient-derived xenograft model of clinical venetoclax/decitabine-relapsed acute myeloid leukemia
In order to determine whether co-inhibition of BCL-2 and MCL-1 could overcome venetoclax resistance in vivo, we developed an AML PDX model using cells from a patient who initially responded to venetoclax/decitabine therapy, but relapsed after three cycles (Figure 6A; Online Supplementary Table S1). PDX-bearing mice were treated with venetoclax, AZD5991, AZD4573, venetoclax plus AZD5991, or venetoclax plus AZD4573 (Figure 6A). All treatments statistically significantly decreased circulating blasts compared to controls (P≤0.0001, treatment day [d] 18). The venetoclax plus AZD5991 or venetoclax plus AZD4573 group had statistically significantly fewer circu- lating blasts than the venetoclax (P<0.01) and AZD5991 (P<0.0001) groups or the venetoclax (P=0.0001) and AZD4573 (P<0.001) groups, respectively (Figure 6B). Analyses of BM samples (treatment d 25) yielded similar results (Figure 6C). Spleens from AZD4573- or venetoclax plus AZD4573-treated mice had a statistically significantly lower leukemia burden compared to controls, which were
haematologica | 2022; 107(1)
67