Downregulation of the Common Cytokine Receptor Subunit Beta c by Omacetaxine in CML: A Potential Molecular Mechanism to Overcome Cytokine-Mediated Resistance against BCR-ABL-Inhibitors.

Inhibition of BCR-ABL by imatinib is standard treatment for patients with chronic myeloid leukemia (CML). Despite favourable response rates, overcoming primary and secondary resistance against BCR-ABL inhibitors is of upmost clinical importance. A proposed mechanism of CML stem-cell survival is cytokine-dependent activation of anti-apoptotic and promitogenic signalling cascades despite continued treatment with tyrosine kinase inhibitors (TKI). Additionally, resistance inducing point mutations of the BCR-ABL kinase domain besides lowering the drug-target affinity of TKIs also seem to interfere with cytokine signalling by not yet fully understood mechanisms. Omacetaxine mepesuccinate (OM, formerly known as homoharringtonine) is showing promising results in phase II clinical trials even in patients with the highly resistant mutation T315I. Preliminary results suggest deselection of the aggressive T315I-clone by OM. We therefore went back to pre-clinical resistance models to study mechanisms of OM-dependent antileukemic activity.

Two murine cell lines with induced expression of unmutated and T315I-mutant BCR-ABL (32Dp210, Baf3p210), and a human CML cell line with induced imatinib-resistance due to continuous drug exposure (KBM5s, KBM5r-T315I) were used in addition to primary CD34+ enriched stem cell cultures.

Dye exclusion proliferation assays confirmed BCR-ABL-dependent sensitization of cell lines to OM in the low nanomolar dose range. Expression of BCR-ABL-mutant T315I does not confer cross-resistance to OM in myeloid cell lines. However, it slightly reduces OM-sensitivity in lymphoid Baf3p210-T315I cells with a 2.5-fold increased IC50-value. OM induced suppression of cell viability is mediated by Caspase-3-dependent apoptosis as detected by flow cytometry. Addition of Interleukin-3 (IL3), shown to revert BCR-ABL-inhibitor dependent cytotoxicity in the murine 32Dp210 and Baf3p210 cell lines, does not affect OM-induced antiproliferative activity. In addition, cytotoxicity of OM against CD34-enriched CML stem cells grown in the presence of a high vs low physiological growth factor mix (GF) is unaffected by the respective GF-condition. Looking at potential mechanisms, we found marked OM-induced downregulation of the beta-subunit of the IL3-receptor (IL3-R) in both, cell lines, and primary stem cell cultures as detected by Western blot, irrespective of the mutational status, at the clinically relevant concentration of 40 nM OM. Due to the significantly reduced IL3-R-expression in BCR-ABL-T315I-expressing cells compared to unmutated BCR-ABL-expressing cells, OM-treatment leads to near complete eradication of the IL3-R in BCR-ABL-T315I positive cells. Combined treatment of Baf3p210 cells with Nilotinib and OM reveals that OM overrides cytokine mediated rescue of TKI treatment.

The observed cytokine-independent in-vitro cytotoxicity of OM may be explained by OM-induced suppression of IL3-R-expression. Loss of IL3-R-expression in BCR-ABL-T315I expressing cells could be one mechanism governing the clinically observed deselection of the resistant clone in patients treated with OM. Cytokine receptor directed action of OM in CML needs to be explored as a potential strategy to overcome cytokine dependent resistance development against TKI-treatment, and to further explore OM-activity against disease maintaining stem cells.

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