Abstract 340: Mitochondrial priming of new targeted agents in acute myeloid leukemia
Authors: Ishizawa Jo , Kojima Kensuke , Duvvuri Seshagiri R. , McQueen Teresa , Ruvolo Vivian R. , Nogueras-Gonzalez Graciela M. , Huang Xuelin , Pierceall William , Cardone Michael , Lena Ryan , Doykan Camille , Shacham Sharon , Kauffman Michael , Konopleva Marina , Andreeff Michael 
Published: May 2015

Abstract
As numerous molecularly targeted agents are entering clinical trials, predictive testing is highly desirable. We investigated if response to certain agents correlates with the recently reported method of BH3 profiling (Chonghaile TN et al, Science, 2011), a functional assay developed by Letai's group that measures tumor cell mitochondrial priming by measuring mitochondrial outer membrane permeabilization (MOMP) following exposure to the peptide-mimicking BH3 domains of BH3-only proteins. Mitochondrial priming has been reported to be correlated with clinical responses to conventional chemotherapy in solid tumors and hematological malignancies (Vo TT et al, Cell, 2012, Pierceall W et al, Mol Cancer Ther, 2013).
Twenty-two AML lines were tested. Cells were permeabilized with digitonin and exposed to standardized doses of BH3 peptides (BIM, PUMA, NOXA, BAD, BMF, HRK, or PUMA2A). JC-1 was used for detection of MOMP and served as a measure of sensitivity to each peptide (reported as %[BH3 peptide]). Also, BCL-2, BCL-XL and MCL-1 expression levels were determined by Western blot. In addition to studies of untreated cells, treatment effects of different anti-leukemia drugs (AraC, Nutlin-3a, KPT-330 [Selinexor] and ABT-199) were determined over a wide dose range and denoted as ([specific apoptosis] = [(%Annexin V+ cells at each dose) - (%Annexin V+ cells at 0 μM)]/[100- (%Annexin V+ cells at 0 μM)]). Mixed linear models were used for analysis. As expected, ABT-199 sensitivity positively correlated with %[BAD]-%[HRK] (|β| = 3.22, p &lt; 0.001), which is compatible with BCL-2 dependency of ABT-199 (while BAD is binding to BCL-2 and BCL-XL, HRK is binding to BCL-XL only and the difference is therefore BCL-2-specific). This was supported by the observed correlation between %[BAD]-%[HRK] and BCL-2 protein expression levels (r=0.619; p = 0.018). AraC sensitivity showed a similar correlation with %[BAD]-%[HRK] (|β| = 1.61, P &lt; 0.05). KPT-330 sensitivity in p53 wild-type cell lines positively correlated with %[PUMA] (|β| = 0.92, P = 0.054), consistent with the notion that KPT-330 induces PUMA through p53 activation. Unexpectedly, Nutlin-3a activity did not correlate with any of the BH3 peptides. Results indicate that ABT-199, KPT-330 and Nutlin-3a show different modes of action in terms of BH3 peptide dependency, supporting potential combination effects of these agents. For ABT-199, increased MCL1 levels were associated with diminished cytotoxicity (r=0.532; p=0.05), as expected. For Ara-C, a similar correlation with MCL-1 was noted (r=0.505; p=0.06), but no correlations were observed for Nutlin-3a and KPT-330.
In conclusion, BH3 profiling is a promising tool to predict the BH3 peptide dependency of the BH3-mimetic ABT-199 and the XPO1-inhibitor KPT-330. Functional BH3-profiling appears to be superior to the static quantitation of Bcl-2 family protein levels in AML. Furthermore, mitochondrial priming may be useful for the rational design of new combination therapies.
Citation Format: Jo Ishizawa, Kensuke Kojima, Seshagiri R. Duvvuri, Teresa McQueen, Vivian R. Ruvolo, Graciela M. Nogueras-Gonzalez, Xuelin Huang, William Pierceall, Michael Cardone, Ryan Lena, Camille Doykan, Sharon Shacham, Michael Kauffman, Marina Konopleva, Michael Andreeff. Mitochondrial priming of new targeted agents in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 340. doi:10.1158/1538-7445.AM2014-340