Ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017): a novel scaffold that resensitizes multidrug resistant leukemia cells to chemotherapy.

The rapid development of drug resistance is a major obstacle in the treatment of cancer. Multidrug resistance (MDR) is a common form of acquired drug resistance in which cancer cells, upon exposure to a single chemotherapeutic drug, develop resistance to a variety of drugs that are structurally and mechanistically unrelated. Due to the rapid development of MDR, many patients do not respond well to chemotherapy and often relapse with a more aggressive form of cancer. Therefore, there is a need for new anticancer agents that can selectively target MDR cancers and ideally re-sensitize them to standard therapies. Cancer cells can acquire MDR through various mechanisms. One major mechanism is the over-expression of ATP-binding cassette (ABC) transporter proteins, such as p-glycoprotein (P-gp).(1–7) Tumor cells with elevated ABC transporter proteins can decrease the intracellular concentration of anticancer drugs through ABC transporter-mediated enhanced efflux, leading to MDR. The other major mechanism is through altering the apoptotic pathway. Apoptosis is a programmed cell death process regulated by a variety of cellular signals. The two main apoptotic pathways are the intrinsic and extrinsic pathways.(8–10) The intrinsic pathway is of particular interests because a majority of chemotherapeutic drugs are believed to rely on this pathway to induce programmed cell death among cancer cells.(11–16) B-cell lymphoma-2 (Bcl-2) family proteins play an important role in the intrinsic apoptosis, consisting of anti-apoptotic members, such as Mcl-1, Bcl-2, and Bcl-XL, and pro-apoptotic members, including Bax, Bad, Bak, Bid, Bim and Noxa. The anti-apoptotic and pro-apoptotic members of the Bcl-2 family proteins antagonize each other to control cytochrome c release from mitochondria and to determine the fate of a cell – death or survival.(17) As key regulators that prevent programmed cell death, the anti-apoptotic Bcl-2 family proteins are over-expressed in 60–90% of all cancers,(18, 19) being considered as one general mechanism by which cancer cells gain resistance against standard therapies. Results of recent studies have revealed that the Bcl-2 family proteins also localize on other cellular compartments besides mitochondria, including the nuclear envelope and endoplasmic reticulum (ER).(20–22) Although the function of the Bcl-2 family proteins on the nuclear envelope is not fully understood, there have been some insights into their role on the ER membrane. Calcium release from the ER can act as a secondary messenger for cell death signaling, which involves multiple communication loops between ER and mitochondria that help regulate apoptosis.(23, 24) The ER membrane-localized Bcl-2 family proteins regulate calcium homeostasis and subsequently, programmed cell death, by interacting with various ER calcium transporters. For instance, Bcl-XL and Bax can interact with and regulate the inositol triphosphate receptors (IP3Rs), which release ER Ca2+ into the cytosol.(25, 26) Some reports also suggest that Bcl-2 interacts with and regulates the function of sarco-endoplasmic reticulum ATPase (SERCA) pumps, which transport cytosolic Ca2+ into the ER.(27–29) Given that the over-expression of the anti-apoptotic Bcl-2 family proteins and dysregulation in Ca2+ homeostasis have both been reported to be responsible for MDR in cancer,(30–33) simultaneously regulating both pathways may provide an effective strategy to overcome drug resistance in cancer. We previously reported the discovery of sHA 14-1 (Figure 1), a dual inhibitor of Bcl-2 and SERCA.(34) Recently, we developed ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) (Figure 1), a more potent analog of sHA 14-1, which demonstrates increased cytotoxicity towards a variety of MDR resistant cancer cell lines as well as synergism with a wide range of standard therapies.(35, 36) The improved potency observed in MDR cancer cell lines, also known as collateral sensitivity (CS), is a relatively rare phenomenon in cancer therapy and the mechanisms leading to CS are largely unknown. Given its synergistic and CS properties, CXL017 harbors the potential to improve the treatment response of cancer patients. Figure 1 The chemical structures of sHA 14-1 and CXL017. In this report, we show that CXL017 demonstrates increased efficacy towards tumors inoculated from HL60/MX2, an MDR cell line, in a xenograft mouse model. Moreover, HL60/MX2 cell line fails to develop stable resistance to CXL017 whereas it develops stable resistance (>2000-fold) to cytarabine (Ara-C) and ABT-737 (a representative Bcl-2 inhibitor). More significantly, upon long-term exposure to CXL017, the HL60/MX2 cell line becomes hypersensitive to standard therapies, such as cytarabine, doxorubicin, mitoxantrone, and etoposide. Molecular characterization of CXL017 treated HL60/MX2 cell lines reveals significant changes among several Bcl-2 family proteins, SERCA proteins, and ER calcium content. These changes may be responsible for CXL017-induced re-sensitization of drug resistant cancer cell lines.