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.

Topics:
apoptosis, chlorambucil, nylons, stem cells, tumor cells, malignant, adverse effects, alkylating agents, annexin a5, cancer, combined modality therapy

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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