Abstract C3: Activating necroptosis in acute myeloid leukemia through inhibition of PKC, calmodulin, and HOX/PBX dimerization

Abstract

Abstract The molecular mechanisms underlying the pathogenesis of acute myeloid leukemia (AML) have been extensively studied, and are known to involve members of the HOX family of transcription factors, both as partners in chimeric fusion proteins, and also in their wild type form. However, the role of HOX proteins in the survival of AML cells has proved difficult to assess as many have redundant functions, which makes a conventional knock down experiment difficult to interpret. An alternative strategy to targeting HOX proteins is to inhibit their interaction with the PBX co-factor, which can be achieved using a short, cell-penetrating peptide (HXR9) that mimics the conserved hexapeptide in HOX proteins responsible for PBX binding. HXR9 has been shown to induce apoptosis in malignant B cells, and a number of AML cell lines. In order to evaluate the molecular mechanisms underlying the cytotoxicity of HXR9 in AML cells, we determined the sensitivity of a number of AML-derived cell lines. Three of these (KG1, HEL 92.1.7, and HL-60) are derived from primary AML, and two from secondary AML (KU812F, and K562). The IC50s of cell killing by HXR9, as determined using a lactose dehydrogenase (LDH) assay, were 4.5, 6.1, 16.9, 9.1, and 10.4 μM, respectively. The K562 cells were also sensitive to HXR9-mediated killing in vivo, as administration of HXR9 could significantly inhibit the growth of K562 flank tumors in a mouse xenograft model. We investigated whether these cells underwent apoptosis after HXR9 treatment. There were no definitive changes associated with apoptosis including caspase-3 activation, PARP cleavage, or nuclear fragmentation. Furthermore, cell death was not dependent on ATP, and could not be reversed by the pan-caspase inhibitor z-VAD-FMK. We therefore explored the possibility of necrotic cell death. Neither K562 nor HL-60 cells could be rescued using CsA, an inhibitor of mitochondrial necrosis that targets the CypD protein. However, inhibition of the RIP1 kinase using its inhibitor Nec-1 resulted in a significant rescue of K562 and HL-60 cells from HXR9-mediated cytotoxicity. RIP1 is a central component of the necroptosis pathway, suggesting that this might play a key role in HXR9-induced cell death. We also explored further molecular pathways that might influence necroptosis. Inhibition of signaling through p38, JNK, and MEK/ERK had no effect on HXR9 cytotoxicity, nor did the inhibition of the p53 tumor suppressor protein, although HXR9 treatment of both K562 and HL-60 cells resulted in a significant increase in expression of the p21 tumor suppressor gene. Furthermore, inhibition of both protein kinase C (PKC) and calmodulin significantly sensitized cells to HXR9. Taken together, our findings point to a mechanism of HXR9-mediated cell death that depends not on apoptosis, but instead on necroptosis, and which can be blocked by PKC signaling. Necroptosis is considered to be a regulated form of necrosis that in some respects parallels apoptosis, as it can be triggered by the same external stimuli. Its interaction with PKC and calmodulin-mediated signaling points to possible synergistic approaches when targeting AML. Citation Format: Raed Alharbi, Ruth Pettengell, Mohamed El-Tanani, Hardev S. Pandha, Richard Morgan. Activating necroptosis in acute myeloid leukemia through inhibition of PKC, calmodulin, and HOX/PBX dimerization. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C3.