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.
Authors: Papa Veronica , Tazzari PierLuigi , Chiarini Francesca , Ricci Francesca , Martinelli Giovanni , Bontadini Andrea , McCubrey James , Lindner Lars H , Eibl Hansjoerg , Martelli Alberto M. 
Published: April 2020

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

Interference with phospholipid turnover and lipid signaling, Induction of stress signaling (SAPK/JNK) and apoptosis, 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+, CD38Low/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.