MI-238 is a novel and potent Mcl-1 inhibitor
As an important pro-survival protein, Mcl-1 is over-expressed in various types of cancer. However, no specific Mcl-1 inhibitor is currently available for clinical use. Through cytotoxicity screening using the paired H1299 parental and Mcl-1 knockout (KO) cells, we discovered a small molecule that selectively inhibited the viability of H1299 parental cells, but not Mcl-1 KO cells (Figure S1), and we named this compound MI-238 (Fig. 1A). MI-238 was docked into Mcl-1 BH3 binding groove and proximal to the BH1 domain of Mcl-1 (Fig. 1A). To directly measure MI-238/Mcl-1 binding, we prepared GST tagged Mcl-1 recombinant protein (Fig. 1B). MI-238 exhibited a Ki of 0.45 ± 0.05 µM to human Mcl-1 protein in FP (fluorescence polarization) assay (Fig. 1C). Meanwhile, Isothermal Titration Calorimetry (ITC) assay also showed that MI-238 binding to GST-Mcl-1 with Kd of 0.23 µM, meanwhile, MI-238 did not bind with GST protein (Fig. 1D, E). Mcl-1 exerts its pro-survival function through binding with BH3-only pro-apoptotic proteins, such as Bak. To test whether MI-238 binding to Mcl-1 could disrupt Mcl-1/Bak association, purified Mcl-1 and Bak proteins were incubated in presence of increasing concentrations of MI-238. As shown in Fig. 1F, MI-238 disrupted Bak/Mcl-1 interaction in a dose-dependent manner in vitro. Collectively, these results demonstrated that MI-238 directly binds to Mcl-1 and inhibits Mcl-1 anti-apoptotic function.
MI-238 selectively induces apoptosis in Mcl-1 proficient cells.
To measure whether MI-238 induced apoptosis depends on Mcl-1, we generated H1299 Mcl-1 KO cells. Knockout of Mcl-1 in H1299 and MEF cells did not significantly affect the expression Bcl-2 protein. However, BH3-only proteins including Bak, Bim and Bax were shifted to Bcl-2 and Bcl-xL in Mcl-1 deficient cells, which indicating Mcl-1 KO cells relied more on Bcl-2 and Bcl-xL for survival (Fig. 2A, C, Figure S2). The Annexin V apoptosis assay revealed that 20 µM of MI-238 treatment induced apoptosis in 50.1 ± 1.1% of parental H1299 cells, but intriguingly, 20 µM MI-238 failed to induce apoptosis in Mcl-1 deficient H1299 cells (Fig. 2B). Cleavage of caspase 3 initiates apoptotic DNA fragmentation and is recognized as an apoptosis hallmark. Consistent with Annexin V assay, MI-238 treatment caused caspase 3 cleavage in H1299 parental cells, but not in Mcl-1 KO cells (Fig. 2E). Similarly, we found that MI-238 treatment also induced apoptosis in wild type (WT) mouse embryonic fibroblast (MEF) cells, but not in Mcl-1 KO MEF cells (Fig. 2D, F). These results demonstrated that MI-238 induced apoptosis is dependent on proficient Mcl-1.
Mi-238 Effectively Kills Aml Cells
Induction of apoptosis through targeting Bcl-2 anti-apoptotic proteins is an effective therapeutic strategy for hematologic malignancies, and Bcl-2 inhibitor venetoclax is widely used to treat CLL and AML. To test the therapeutic efficacy of MI-238 in AML cells, we treated a variety of AML cells with increasing concentrations of MI-238 and found that the IC50 of MI-238 against AML is around 5–30 µM (Fig. 3A). In addition, the IC50 of MI-238 was inversely proportional to the Mcl-1 protein levels among AML cells (Fig. 3B, C). We then examined the PARP1 and caspase 3 cleavage, the well-known apoptosis markers in Molm13 and MV-4-11 cells after MI-238 treatment . As shown in Fig. 3D, MI-238 induced PARP1 and caspase 3 cleavage in a dose-dependent manner, which indicating MI-238 potently induces apoptosis in AML cells. We further employed Annexin V staining to measure the apoptosis frequency in Molm13 and MV-4-11 cells after MI-238 treatment, and found that 40 µM of MI-238 caused 60 ± 0.4% and 35 ± 1.5% apoptotic cell death in Molm13 and MV-4-11 cells respectively (Fig. 3E, F). Taken together, these results demonstrated that MI-238 effectively induces apoptosis in AML cells.
Mi-238 Treatment Induces Activation Of Bh3-only Proteins
Mcl-1 inhibits apoptosis through sequestering pro-apoptotic BH3-only proteins, such as Bax, Bak, Bim and Puma. To check whether MI-238 treatment could release BH3-only proteins from Mcl-1, we performed immunoprecipitation (IP) assay using antibodies against anti-apoptotic proteins including Mcl-1, Bcl-2 and Bcl-xL, and the result showed that Mcl-1 mainly binds to Bak, Bim, and Puma, but not Bax in Molm13 cells (Fig. 4A). Meanwhile, treatment of MI-238 could disrupted Mcl-1 association with BH3-only pro-apoptotic proteins including Bak, Bim and Puma (Fig. 4A). Whereas, MI-238 failed to interrupt the binding of BH3-only proteins to Bcl-2 and Bcl-xL (Fig. 4A), suggesting MI-238 treatment specifically inhibits Mcl-1, but not Bcl-2 and Bcl-xL. Bak release from Mcl-1 causes its conformation change and homo-oligomerization to initiate apoptosis. We then employed flow cytometry analysis of Bak activation after MI-238 treatment by staining with activation-specific antibody. Consistent with IP assay, MI-238 treatment induced Bak activation in Molm13 cells in a dose-dependent manner (Fig. 4B).
Activation of BH3-only proteins results in release of cytochrome C from mitochondria into the cytosol, which in turn triggers apoptosis. We then performed cell fractionation analysis to examined cytochrome C level in mitochondrial and cytosol after MI-238 treatment. As shown in Fig. 4C, we observed a significant decrease of mitochondrial cytochrome C level and increase cytosol cytochrome C after MI-238 treatment, indicating MI-238 could induce cytochrome C translocation from mitochondrial to cytosol.
Mi-238 Synergizes With Venetoclax To Induce Apoptosis In Aml Cells
Given that Mcl-1 is the primary venetoclax resistant protein, we then tested whether MI-238 could sensitize AML cells to venetoclax treatment. As shown in Fig. 5A-B, 10 µM of MI-238 induced 34.8 ± 1.2% apoptosis, 0.02 µM of venetoclax caused 26.1 ± 1.3% apoptosis in Molm13 cells,while, their combination induced 87.4 ± 0.3% apoptosis. Besides, synergistic effects of MI-238 and venetoclax on apoptosis induction in Molm13 cells have also been detected on different combinations (10µM + 0.1µM, 5µM + 0.02µM, and 5µM + 0.1µM). (Fig. 5C, D, E, F and Figure S3). Consistent with annexin V staining assay, combination treatment of MI-238 and venetoclax induced greater caspase 3 cleavage compared with treatment of MI-238 only or venetoclax only (Fig. 5G). In addition, MI-238 and venetoclax combination induced significantly greater activation of Bak compared with MI-238 or venetoclax treatment alone (Figure S4). These results indicate that MI-238 could sensitize AML cells to venetoclax treatment and combination of MI-238 and venetoclax induces synergistic anti-tumor effects.
Mi-238 And Venetoclax Have A Synergistic Effect In Aml Xenografts
To evaluate the therapeutic efficacy of MI-238 and venetoclax in vivo, mice were intravenously (i.v) injected with Molm13 cells stably expressing luciferase (Molm13-Luc) to generate Molm13 AML xenograft model. We started treatment at 10 days after cell implantation, and monitored the cancer progression once a week by the bioluminescence imaging (Fig. 6A). At the beginning of the treatment (10 days after Molm13 implantation), we clearly detected luciferase signal in all mice, that is proportional to amount of leukemic cells (Fig. 6B). Bioluminescence images obtained after drug treatment (17 and 24 days) showed a significant reduction of leukemia burden in response to MI-238 alone, while, the greater suppression of leukemia progression was seen in the combination treatment compared with MI-238 or venetoclax alone (Fig. 6B).
Meanwhile, the percentage of Molm13 cells in the murine peripheral blood was quantified by flow cytometry using anti-human CD45 (hCD45) and anti-hCD33 monoclonal antibodies, since hCD45/hCD33 double positive was recognized as the human AML marker. As shown in Fig. 6C and D, MI-238 alone treatment significantly reduced the hCD45+/hCD33 + cells in the peripheral blood compared with vehicle-treated mice (11.7 ± 3.4% vs. 24.7 ± 3.9%). Although, venetoclax alone also decreased percentage of hCD45+/hCD33 + cells (10.5 ± 2.1%), venetoclax in combination with MI-238 could decrease hCD45+/hCD33 + leukemia cells to 3.0 ± 1.7% (Fig. 6C, D, Figure S5). Similarly, immunohistochemical (IHC) analysis of hCD45 + cells in bone marrow also proved that MI-238 treatment alone could significantly decrease tumor burden, while MI-238 in combination of venetoclax induced greater reduction in the tumor burden (Fig. 6E, F). In addition, survival analysis revealed that MI-238 alone or in combination with venetoclax could significantly prolong survival of tumor bearing mice (vehicle treated mice = 24.6 days, vs MI-238 treated mice = 28 days, vs venetoclax treated mice = 29.8 days, vs combination treated mice = 35.8 days) (Fig. 6G).
Mi-238 Treatment Alone Or In Combination With Venetoclax Is Effective In Aml Patient Samples
In order to further validate the therapeutic efficacy of MI-238 and its combination with venetoclax, primary patient AML cells were analyzed. Mononuclear bone marrow cells from 3 different AML patients were treated with increasing concentrations of MI-238 and the apoptosis were analyzed by annexin V staining. Consistent with AML cell line, MI-238 treatment induced apoptosis in AML patient samples in a dose-dependent manner and more than half of bone-marrow mononuclear cells underwent apoptosis in presence of 40µM MI-238 treatment in all three patient samples (Fig. 7A, B, C, FigureS6). Similarly, we detected increasing cleavage of caspase 3 after treatment of MI-238 (Figure D, E, F), which confirmed that MI-238 potently induced apoptotic cell death in tumor cells from AML patient samples. Meanwhile, 20µM of MI-238 treatment failed to induce apoptosis in bone-marrow mononuclear cells from healthy donor (Figure S7). Then, we treated patient AML cells with MI-238, venetoclax or their combination to test whether MI-238 could sensitize AML patient samples to venetoclax. As shown in Fig. 7G-I, we detected significantly greater apoptosis in patient AML cells treated MI-238 plus venetoclax compared with cells treated MI-238 or venetoclax alone. Besides, greater cleavage of caspase 3 was detected in patients AML cells treated with MI-238 and venetoclax combination, which further demonstrated that MI-238 is effective in primary patient AML cells (Fig. 7J, K, L). Besides, a significantly synergistic effects of MI-238 and venetoclax on apoptosis induction in primary AML patient samples (Fig. 7M, N, O). Collectively, these data demonstrated that MI-238 alone or its combination with venetoclax efficiently induce apoptosis in the bone marrow samples of AML patient, further supporting its therapeutic efficacy to treat AML.