Activity of omacetaxine mepesuccinate against ponatinib-resistant BCR-ABL-positive cells

To the editor:

Abelson murine leukemia viral oncogene homolog (ABL) tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, and dasatinib have improved the survival of Philadelphia chromosome (Ph)-positive leukemia patients.¹  However, despite the impressive efficacy of these agents, disease relapse has been frequently observed. Mutations in the breakpoint region cluster protein (BCR)-ABL kinase domain can cause ABL TKI-resistance.²  One particular BCR-ABL kinase domain mutation, T315I, is associated with a high level of resistance to all available ABL TKIs. Ponatinib (AP24534) is a multitarget TKI. Recently, in the Ponatinib Ph+ Acute Lymphocytic Leukemia (ALL) and Chronic Myeloid Leukemia (CML) Evaluation (PACE) trial, ponatinib showed significant efficacy against Ph-positive leukemia in patients with multiresistant T315I mutations.³  However, in some patients, especially those with Ph-positive ALL, ponatinib-resistant clones were identified.

Omacetaxine mepesuccinate (OM), formally known as homoharringtonine, is a natural alkaloid obtained from various Cephalotaxus species. OM is a first-in-class cephalotaxine in clinical development as an antileukemic therapy. OM acts by binding to the A-site cleft of ribosomes, thereby transiently inhibiting protein synthesis. OM was approved for the treatment of adult patients with chronic- or accelerated-phase CML that was resistant to other therapies.⁴ 

We investigated the efficacy of OM against ponatinib-resistant Ph-positive cells. Ba/F3 ponatinib-resistant (Ba/F3 ponatinib-R) cells have 3 BCR-ABL point mutations (Y253H, E255K, and T315I; data not shown). With 72 hours of OM treatment, the cell growth of Ba/F3 ponatinib-R and Ph-positive ALL cell lines was significantly reduced, even when treated with low concentrations of OM (Figure 1A and supplemental Figure 1A). This agent is also effective against other hematological malignancies such as acute myeloid leukemia.⁵  In contrast, Ba/F3 ponatinib-R cells were resistant to ponatinib (Figure 1B). With 48 hours of treatment, OM-dependent apoptosis was increased (Figure 1C). The resistance of Ba/F3 cells bearing these 3-point mutations to ponatinib is consistent with the high-level resistance conferred by compound mutations such as E255V/T315I,^6,7  although Ba/F3 ponatinib-R cells did not display increased antiapoptotic proteins.

We also examined intracellular signaling. The phosphorylation of BCR-ABL and a downstream molecule, v-Crk avian sarcoma virus CT10 oncogene homolog-like (Crk-L), were decreased (Figure 1D). Protein expression levels of BCR-ABL and Crk-L were also decreased. However, caspase 3 and cleaved Poly (adenosine 5'-diphosphate-ribose) polymerase (PARP) levels were significantly increased in low concentration (Figure 1D). In a previous study, OM was shown to induce apoptosis in leukemic cells because of a selective decrease in short-lived proteins.^8,9  We found that OM treatment reduced the expression of BCR-ABL, as well as heat shock protein (HSP)90, which stabilizes the BCR-ABL protein. We also found that OM reduced the expression level of the antiapoptotic protein Bcl-2. The level of c-Myc expressed was also reduced. We then examined ponatinib-resistant primary Ph-positive ALL and chronic-phase CML samples. The ponatinib-resistant primary cells had several BCR-ABL point mutations (eg, Q252H, E255K/V, and T315I). We found that the growth of primary cells was not affected by ponatinib but was reduced after OM treatment. Similar signaling events occurred in OM-treated primary ALL cells (Figure 1E and supplemental Figure 1B-C). Next, we used the pan-caspase inhibitor Z-VAD-fmk for the inhibition of caspases. Z-VAD-fmk treatment inhibited OM-induced apoptosis but did not prevent the degradation of BCR-ABL, HSP90, and Bcl-2 in Ba/F3 ponatinib-R cells (supplemental Figure 1D). Thus, BCR-ABL, HSP90, and Bcl-2 downregulation was independent of caspase activity.

OM is an inhibitor of protein synthesis. Because OM inhibits the BCR-ABL, Bcl-2, and HSP90 pathways in BCR-ABL-positive leukemia cells by reducing the levels of these proteins, OM has antitumor activity and promotes apoptosis. Our findings suggest that OM may be beneficial in the treatment of leukemia patients with BCR-ABL mutant cells, helping in overcoming ponatinib resistance.