The Polyamide-Chlorambucil Conjugate 1R-Chl Effectively Inhibits Proliferation and Induces Apoptosis in CML Progenitor Cells

Abstract

Imatinib mesylate (IM) is highly effective in the treatment of chronic myelogenous leukemia (CML). However, resistance of malignant progenitors to elimination by IM remains a clinical challenge and new strategies to improve elimination of residual primitive CML cells are required. The polyamide-chlorambucil conjugate 1R-Chl has been developed with the rationale that coupling to a polyamide will increase the DNA sequence specificity of the alkylating agent, and decrease unwanted side effects while retaining the ability to kill cancer cells. In previous studies 1R-Chl was shown to inhibit growth of K562 CML cells in vitro and in vivo (Mol Cancer Ther. 2008; 7:769). Histone H4c, which is highly expressed in a wide range of cancer cells, was identified as a gene target of 1R-Chl. H4c downregulation was associated with chromatin decondensation, possibly exposing otherwise hidden 1R-Chl binding sites. The purpose of the current studies was to investigate whether 1R-Chl could effectively induce apoptosis and inhibit growth of primary CML hematopoietic progenitors. CD34+ progenitors obtained from untreated CML patients and from healthy donors were cultured for 96 hours in growth factor supplemented medium in a range of concentrations of 1R-Chl (0–1μM), its inactive stereoisomer 1S-Chl (0–1μM), and with 5μM IM for comparison. Cells were labeled with CFSE prior to culture and with Annexin-V at culmination of culture to allow flow cytometry assessment of the effects of drug exposure on cell proliferation and apoptosis. In addition CD34+ cells incubated under the same conditions were plated in methylcellulose progenitor culture to assess effects on colony forming cell (CFC) growth. 1R-Chl treatment resulted in significantly increased apoptosis of CML CD34+ cells (from 5.0±1.2% [control] to 41.2±8.0% ([1μM 1R-Chl], n=6, p=001). In contrast, 1S-Chl (1μM) or IM (5μM) treatment did not significantly increase CML CD34+ cell apoptosis. Importantly 1R-Chl, 1S-Chl and IM treatment did not significantly increase apoptosis in normal CD34+ cells (n=3). Combined treatment with 1R-Chl and IM did not result in increased apoptosis of CML CD34+ cells compared to 1R-Chl alone (n=3). Analysis of CFSE fluorescence indicated that 1R-Chl treatment induced dose dependent reduction in proliferation of CML CD34+ progenitors (proliferation relative to untreated cells of 0.39±0.1 with 1μM 1R-Chl, n=4–6, p=.01) with an IC50 value of 0.4μM. The antiproliferative effects of 1R-Chl were significantly greater than those of 1S-Chl (0.6±0.2, n=4–6, p=.02) and IM (0.6±0.1, n=4–6, p=.03). Minor antiproliferative effects were observed in normal controls (0.8±0.1, n=3, p=.0005 [1μM 1R-Chl]; 0.9±0.1, n=3, p=.008 [1μM 1S-Chl] and 0.98±0.1, n=3, p=NS [5μM IM]). The combination of IM and 1R-Chl did not result in further reduction in proliferation of CML CD34+ cells compared to 1R-Chl alone (n=3). A trend towards an increased proportion of undivided cells was observed in response to 1R-Chl, 1S-Chl and IM treatment (from 1.5±0.5% [control] to 19.1±8.7%, 4.4±1.9%, and 11.3±6.7%, n=4–6, respectively). CML CFC growth was suppressed by 96.0±1.5% by 1μM 1R-Chl (n=4–5, p<.001) with an IC50 value of 0.25μM. In comparison, 1S-Chl did not significantly inhibit CML CFC growth and IM suppressed CFC CML growth by 76.1±8.9% (n=4–5, p<.001). 1R-Chl (1μM) suppressed normal CFC growth by 59.3±13.9% (n=3, p=.005, IC50=0.88μM), whereas 1S-Chl and IM did not significantly inhibit normal CFC growth. In summary we observed that the alkylating agent-polyamide conjugate 1R-Chl exerted significant and selective pro-apoptotic and antiproliferative effects on CML CD34+ progenitors. The pro-apoptotic effects of 1R-Chl were more potent than those observed with IM, although as with IM treatment, non-dividing CML progenitors were not eliminated. We conclude that 1R-Chl demonstrates considerable activity against primary CML progenitors and that further studies of its potential role in CML therapy are warranted.