Smac mimetic induces cell death in a large proportion of primary acute myeloid leukemia samples, which correlates with defined molecular markers

Abstract

Apoptosis is deregulated in most, if not all, cancers, including hematological malignancies. Smac mimetics that antagonize Inhibitor of Apoptosis (IAP) proteins have so far largely been investigated in acute myeloid leukemia (AML) cell lines; however, little is yet known on the therapeutic potential of Smac mimetics in primary AML samples. In this study, we therefore investigated the antileukemic activity of the Smac mimetic BV6 in diagnostic samples of 67 adult AML patients and correlated the response to clinical, cytogenetic and molecular markers and gene expression profiles. Treatment with cytarabine (ara-C) was used as a standard chemotherapeutic agent. Interestingly, about half (51%) of primary AML samples are sensitive to BV6 and 21% intermediate responsive, while 28% are resistant. Notably, 69% of ara-C-resistant samples show a good to fair response to BV6. Furthermore, combination treatment with ara-C and BV6 exerts additive effects in most samples. Whole-genome gene expression profiling identifies cell death, TNFR1 and NF-κB signaling among the top pathways that are activated by BV6 in BV6-sensitive, but not in BV6-resistant cases. Furthermore, sensitivity of primary AML blasts to BV6 correlates with significantly elevated expression levels of TNF and lower levels of XIAP in diagnostic samples, as well as with NPM1 mutation. In a large set of primary AML samples, these data provide novel insights into factors regulating Smac mimetic response in AML and have important implications for the development of Smac mimetic-based therapies and related diagnostics in AML.

Keywords: IAP proteins; acute myeloid leukemia (AML); apoptosis; gene expression profiling (GEP); smac mimetic.

Figure 1. Primary AML sample viability after 24 hours of treatment with either BV6 or ara-C

(A) 60 primary AML samples and 6 healthy donor samples, treated with ara-C; mean and SD of the three different ara-C response groups are shown (color code independent of BV6 response), which were labeled sensitive (n = 24), intermediate, i.e. moderate response (n = 11), and resistant (n = 25). The healthy donor samples were classified as resistant to ara-C treatment. (B) 67 primary AML samples and 6 healthy donor samples, treated with BV6; mean and SD of the five different BV6 response groups defined by their respective viability at 1 and 10 μM BV6 (indicated by arrows) are shown; 26 samples were labeled BV6-sensitive, 8 were sensitive to low intermediate, 14 were low intermediate, 10 were high intermediate, and 9 samples were labeled BV6-resistant. The healthy donors were classified as high intermediate to resistant. (C) Classification of 67 AML samples into 3 different BV6 response groups, according to their individual sensitivity: sensitive (n = 26 + 8 = 34), low intermediate (n = 14, later excluded in further analyses), and resistant (n = 10 + 9 = 19) cases.

Figure 2. Viability after 24 hours of combination treatment of ara-C and IAP inhibitor BV6 in primary AML samples

Combination of ara-C (increasing doses as indicated) with BV6 (single dose: 2 μM), according to the respective ara-C response group (sensitive (A, B), intermediate (C, D), resistant (E, F), see also Figure 1) and the achieved effect of the combination (more than additive (A, C, E) or additive and less than additive (B, D, F)). Mean and SD of all samples in the respective group (sample size as indicated in each graph) are shown, with the three curves representing ara-C single treatment, combination of ara-C with a constant dose of 2 μM BV6 (measured ara-C+BV6), and the theoretical viability of this combination, as defined by the Bliss independence rule (theoretical ara-C + BV6).

Figure 3. Comparison of gene expression levels of several apoptosis-relevant genes in BV6-sensitive (n = 26) vs. -resistant (n = 14) cases, measured by qRT-PCR in 40 primary AML samples

All expression levels were normalized to ACTB expression levels. Shown are individual expression values as scatter plot, with closed symbols for BV6-sensitive and open symbols for BV6-resistant samples, as well as group median and statistical test results (Mann-Whitney U test; *P < 0.05; ns = non-significant, P ≥ 0.05).