Erucylphosphohomocholine, the First Intravenously Applicable Alkylphosphocholine, Induces Cell Cycle Arrest, Apoptosis, and Synergizes with Chemotherapeutic Drugs in Acute Myelogenous Leukemia Cells-a Novel Therapeutic Approach for Leukemia.

Alkylphosphocholines are promising new drugs for cancer treatment. They kill cancer cells by evoking multiple signaling pathways:

1. Interference with phospholipid turnover and lipid signaling,

2. Induction of stress signaling (SAPK/JNK) and apoptosis,

3. Inhibition of survival and proliferation pathways (PI3K/AKT, MEK/ERK).

We have investigated the therapeutic potential of the novel alkylphosphocholine, erucylphosphohomocholine (ErPC₃), on human acute myelogenous leukemia (AML) cells. At variance with other alkylphosphocholines, such as perifosine, ErPC₃ can be administered i.v. as it does not cause hemolysis. ErPC₃ was tested on THP-1 cells (which display activation of both PI3K/AKT and MEK/ERK pathways), and on HL60 and NB4 cell lines (which only have MEK/ERK activation), as well as primary AML cells. THP-1, HL60, and NB4 cells have a non-functional p53 pathway. At short (6 h) incubation times, the drug blocked AML cells in G2/M phase of the cell cycle, whereas at longer incubation times (24 hr), it decreased survival and induced cell death by apoptosis. The IC₅₀ for ErPC₃ with THP-1 cells was 5.0 μM, whereas with HL60 and NB4 cells it was 10.0 μM. In THP-1 cells, ErPC₃ caused Akt dephosphorylation on Ser 473 and Thr 308, however it also downregulated total Akt expression levels. Downregulation of total Akt levels was blocked by a caspase-3 inhibitor. At 3 μM ErPC₃, induced a complete dephosphorylation of ERK 1/2, whereas a sizable MEK1 dephosphorylation was seen only at 10 μM. The protein phosphatase inhibitor okadaic acid blocked ERK 1/2 dephosphorylation induced by ErPC₃, suggesting dephosphorylation was due to increased phosphatase activity. ErPC₃ activated JNK 2 (54- kDa), and a JNK 1/2 peptide inhibitor markedly reduced ErPC₃-elicited apoptosis in a dose-dependent manner. ErPC₃ did not significantly affect the expression of proteins which are involved in mitochondrial control of apoptosis, including Bax, Bcl-XL, Mcl-1, AIF, Bcl2, p-Bcl2, and survivin. Only Puma expression was downregulated. ErPC₃ activated apical caspases −2, −8, −9, and −10, as well as the executioner caspase−3. Pharmacological inhibitors of either caspase−3 or −9 completely blocked ErPC₃-induced apoptotic cell death. ErPC₃ synergized with etoposide (CI:0.16), doxorubicin (CI: 0.48), and mitoxantrone (CI: 0.33) when the drugs were administered together. In contrast, when the chemotherapeutic drugs were administered prior to ErPC₃, synergism was detected with mitoxantrone (CI: 0.67) and etoposide (CI: 0.15), whereas combinations with doxorubicin resulted in antagonism (CI:1.71). If ErPC₃ was administered prior to the chemotherapeutic drugs, synergism was detected with doxorubicin (CI: 0.79) and etoposide (CI: 0.40), but not with mitoxantrone (CI: 1.41). Moreover, ErPC₃ was cytotoxic for AML blasts with activated PI3K/Akt and MEK/ERK pathways (IC₅₀: 10 μM at 72 h) and for AML primary cells with only activation of MEK/ERK signaling (IC₅₀: 13 μM at 72 h). Remarkably, ErPC₃ induced a significant apoptosis (30–40%) of primary blast cells, especially in the compartment (CD34₊, CD38^Low/Neg, CD123⁺) enriched in putative leukemic stem cells. This observation was also supported by the cytotoxic effects of ErPC₃ on AML blasts displaying high aldehyde dehydrogenase activity. ErPC₃ also reduced the clonogenic activity of CD34⁺ cells from AML patients displaying constitutive PI3K/Akt and/or MEK/ERK upregulation, but not CD34⁺ cells from healthy donors. Our findings indicate that ErPC3, either alone or in combination with existing drugs, is a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K/Akt and/or MEK/ERK survival pathways, even in the absence of a functional p53.