Leukemia & Lymphoma

Induction of apoptosis by MCL-1 inhibitors in chronic lymphocytic leukemia cells

Lisa S. Chen, Michael J. Keating, William G. Wierda & Varsha Gandhi
BCL-2 family proteins play a critical role in the survival of chronic lymphocytic leukemia (CLL) cells and a number of new agents have been developed to target pro-survival protein members of this family. While there are six members of the BCL-2 anti-apoptotic family, BCL-2, BCL- XL, and MCL-1 are the main candidates that have been shown to play functional roles in CLL biology. BCL-2 and BCL-XL are targeted by navitoclax, which has been used both in preclinical and clinical setting for CLL . Due to BCL-XL-targeted undesired impact of navitoclax on platelet numbers, BCL-2-selective molecule, ABT-199 (venetoclax), was developed. ABT-199 demonstrated effective- ness preclinically and clinically and is now approved for the treatment of patients with relapsed CLL . In contrast to targeting BCL-2 and BCL-XL, direct MCL-1 inhibitors have not been reported in the setting of CLL.

The role of MCL-1 appears to be crucial in CLL in particular and is associated with therapeutic resistance and clinical outcome . Recent investigations have reported that a resistance mechanism to ABT-199 may be increased MCL-1 protein levels . This emphasizes the therapeutic utility of MCL-1 inhibition given the emergence of patients developing resistance and experiencing disease progression.

Mechanistically, the MCL-1/ NOXA axis has been key in drug strategies that elicit apoptosis induction, thus inhibition of MCL-1 is a potential strategy for CLL. MCL-1 has been described as an ‘undruggable’ target, due to its comparatively more rigid electropositive binding groove as well having high affinity with its BH3-only protein binding partners . Previous investigations of MCL-1 inhibition included methods that affected protein expression indirectly, such as downregulation of MCL-1 transcript and protein levels by CDK inhibitors and anthracyclines . Other approaches that directly bind MCL-1 such as BH3 peptides and small molecules for target inhibition were insufficiently specific, leading to off-target effects.

One of the first direct small molecule MCL-1 inhibitors reported was BH3-mimetic A-1210477 (Ki 0.45 nM), which was identified using high-throughput screening and structure-guided design . Induction of apoptosis was observed in hematological and solid tumor cell lines in vitro, and treated cells exhibited classic markers of apoptosis such as PARP cleavage, cytochrome C release, and disruption of mitochondrial membrane potential . A-1210477 activity was associated with disruption of MCL-1:BIM protein complex binding, increased BAK protein levels, and functional activation. Increased MCL-1 protein levels were also reported with A-1210477 that may be due to reduced MCL-1 binding with NOXA and MULE, which mediate proteasomal degradation of MCL-1 . Although A-1210477 has been investigated in human cell lines previous investigations have not been reported in primary CLL cells thus far.

We first evaluated the impact of A-1210477 in vitro in primary cells from peripheral blood of patients with CLL to determine whether MCL-1 inhibition results in induction of cell death. ABT-199, a bona fide CLL agent that specifically inhibits BCL-2 was used as a control comparator for apoptosis induction in primary CLL cells. After 24 h of A-1210477 exposure, there was a concentration-dependent induction of apoptosis as determined by increased annexin-V positivity measured by flow cytometry. Data from these eight patients also suggest heterogeneity in apoptotic response that was also evident with ABT-199.

The mean of annexin-positive cells was 34%, 44%, and 66% with 3, 10, and 30 lM A- 1210477, respectively, compared with 26% in cells treated with vehicle DMSO alone . The results with 10–30 lM A-1210477 or 5–10 nM ABT-199 were statistically significant . The levels of caspase activation were similar with the average percent- age of cleaved caspase 3/7 positive cells measured as 33%, 45%, and 60% with 3, 10, and 30 lM A-1210477

Biological and molecular effect of MCL-1 inhibitor A-1210477 in primary cells from patients with CLL.

Selectivity of A-1210477 against BCL-2 family proteins.
percentage of annexin-V positive CLL cells cultured with vehicle DMSO alone or 3, 10, 30 lM of A-1210477 or 5–10 nM ABT-199 for 24h.Cells from peripheral blood of eight patients were cultured and analyzed by flow cytometry after staining with annexin.
Mean and SEM levels of apoptosis induction
Statistical significance (ωp < .05) was determined using a two-tailed paired t-test (GraphPad Prism software).
Immunoblot analysis of apoptosis-related and BCL-2 family proteins in CLL cells from two patients treated in vitro as described.
Supplemental , n ¼ 8 With 30 lM A- 1210477, the levels of annexin-positivity and caspase activation were similar with treatment with 5–10 nM ABT-199 for 24 h, and there was a strong correlation between annexin-positivity and caspase 3/7 cleavage Supplemental.

There were two patients with a 17p deletion Supplemental , and the lowest levels of apoptosis induction were measured in the cells from one of these patients (CLL#141), ranging from 17% to 29% annexin-positivity with 3–10 lM A-1210477 after 24 h. Of note, the levels of apoptosis induction from DMSO alone was also the lowest in the CLL cells from these two patients, measured at 6% and 17% compared with the range of 26–35% in the CLL cells from the other six patients .

Immunoblot analysis of CLL cells treated with A- 1210477 also confirmed induction of apoptosis, and both PARP and caspase 3 protein cleavage were detected with 3–10 lM A-1210477 or 10 nM ABT-199 after 24 h . Similar to prior reports in other cell types, we observed an increase in MCL-1 protein levels in CLL lymphocytes; this was more pronounced at the lower concentrations of A-1210477 exposure that correlates with the lower levels of apoptosis measured since higher levels of cell death may associate with decreased MCL-1 protein levels due to degradation. In contrast to MCL-1, protein levels of BCL-2 and BCL-XL remained largely unchanged, which is consistent given the relatively long half-lives of both proteins.

We also evaluated A-1210477 in primary cells from patients with CLL on ibrutinib therapy. Previous reports have suggested a decline in MCL-1 protein in CLL cells from patients on ibrutinib therapy and thus we investigated whether prior treatment with ibrutinib would sensitize cells to MCL-1 inhibition. CLL cells from patients on ibrutinib therapy were cultured in vitro with 3–10 lM A-1210477 or 10 nM ABT-199, and after 24 h the levels of apoptosis induction were similar as in lymphocytes from patients not receiving ibrutinib therapy (n ¼ 10, Supplemental). The mean of annexin-positive cells was 20%, 27%, and 52% with 3, 10, and 30 lM A-1210477, respectively, compared with 26% in cells treated with vehicle DMSO alone.

The levels of annexin-positivity were 53% with 5–10 nM ABT-199 after 24 h, which was similar to levels achieved with 30 lM A-1210477. Besides weak affinity, another limitation of A-1210477 is reduced bioavailability due to binding to serum proteins , which is consistent with the micromolar concentrations required to induce apoptosis in primary CLL MCL-1 INHIBITION IN CLL 3.

Biological and molecular effect of MCL-1 inhibitor S63845 at nanomolar concentrations in primary cells from patients with CLL.

(A) Selectivity of S63845 against BCL-2 family proteins.

(B) percentage of annexin-V positive CLL cells cultured with 10, 30, or 100 nM S63845. Cells from peripheral blood of seven patients were cultured and analyzed by flow cytometry after staining with annexin.

(C) Mean and SEM levels of apoptosis induction shown in (B). Statistical significance ωp < .05) was determined using a two-tailed paired t-test.

(D) Immunoblot analysis of apoptosis-related and BCL-2 family proteins in CLL cells from four patients treated as described in (B).

Cells as well as in previous cell line reports. Next generation MCL-1 inhibitor S63845 with greater affinity was developed by Servier and Vernalis. Interestingly, cellular activity was not affected by serum concentration and S63845 was shown to be specific on MCL-1 over BCL-2 and BCL-XL (MCL-1 Ki < 1.2 nM.

In a side-by-side comparison with S63845, the Ki measured for A-1210477 was 28 nM , which was greater than in the initial report . Nanomolar levels were sufficient to induce cell death in various hematological cancer cell lines, and in vivo studies using mouse cancer models demonstrated tumor volume reduction in mice treated with S63845.

Studies in CLL cells have not been reported, thus we obtained S63845 under a material transfer agreement and investigated its impact on primary CLL cells in vitro. Following incubation for 24h, the levels of apoptosis induction increased on average by 17% with 10 nM S63845 over DMSO alone. Apoptosis levels further increased to 30% and 50% with 30 and 100 nM S63845 and all were statistically significant .

There was heterogeneity in patient characteristics of the CLL cells evaluated with S63845 and included patients with trisomy 12, 13q, 11q, and 17p deletion, and both mutated and unmutated IGHV status. Induction of apoptosis was also further confirmed by immunoblot analysis, and both PARP and caspase 3 protein cleavage were detected with0 and 30 nM S63845 after 24 h in cells from four patients .

There was also an increase in MCL-1 protein levels in the cells from the patient with 17p deletion (CLL #152) where lower levels of annexin-V positivity were measured. There was not a substantial change in MCL-1 levels in the other three patients which may be due to the relatively higher levels of apoptosis, leading to caspase-mediated cleavage of MCL-1 protein. Our results demonstrate that MCL-1 inhibition by S63845 can potently induce cell death in CLL cells.

Although A-1210477 and S63845 are not viable candidates for clinical application, our results demonstrate the utility of targeting MCL-1 in CLL cells. Comparison with ABT-199 further elucidates that S63845 is effective at nanomolar levels, which is physiologically achievable. A related derivative of S63845, clinical candidate S64315 (also named MIK665), is currently in a Phase I study for AML/MDS (NCT02979366) and lymphoma and multiple myeloma (NCT02992483), and other MCL-1 inhibitors such as AZD5991 and AMG176 are also in Phase I clinical trials for hematological malignancies.

As described earlier, increased levels of MCL-1 is associated with resistance to ABT-199, which provides rationale for potential strategies combining BCL-2 inhibition with MCL-1 inhibitors. Furthermore, it has been shown before that levels of MCL-1 are high in CLL cells that are resident in the lymph nodes, suggesting that these could benefit with 4 L. S. CHEN ET AL. MCL-1 antagonists .

The results of the MCL-1 inhibitor trials will provide important insight into the utility of MCL-1 inhibition in CLL as monotherapy and the possibility of using such agents in A-1210477 combination with existing approved treatments for CLL.

Funding
This work is supported by the MD Anderson CLL Moon shot program.

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