(M1530-02-11) Circumvention of Targeted Anticancer Drug Resistance by Standardized Yunzhi Extract

Purpose: Coriolus versicolor (commonly known as Yunzhi) is a medicinal mushroom with a long history of use for promoting health and longevity in China and other Asian countries. They are popular supplement taken by the general public to boost the immunity and by cancer patients along with their anticancer therapies. The combination of Yunzhi products with chemotherapeutic drugs has demonstrated promising anticancer effect in both preclinical and clinical investigations. However, the mechanisms contributing to the beneficial therapeutic effect remain elusive. Despite the recent advances in novel anticancer drug discovery, cancer remains a deadly disease with limited treatment options. While the discovery of numerous targeted anticancer drugs represents major breakthroughs in cancer treatment, most patients eventually relapse and develop resistance to the treatment. This study investigated the potential circumvention of drug resistance to two representative targeted chemotherapeutic drugs (osimertinib – EGFR tyrosine kinase inhibitor and olaparib – PARP inhibitor) in non-small cell lung cancer (NSCLC) and acute myeloid leukemia (AML), respectively. Furthermore, as lung cancer is highly heterogeneous, it may be possible to identify specific patient subpopulation(s) who may benefit more from osimertinib+Yunzhi combination according to the data obtained in different NSCLC cell lines harboring specific oncogenic abnormalities.

Methods: Standardized Yunzhi extract, with full characterization of its polysaccharide content and other active ingredients, was purchased from a reliable commercial source. The circumvention of osimertinib or olaparib resistance was studied in a panel of NSCLC and AML cell lines displaying different osimertinib/olaparib resistance mechanisms, respectively. The colorimetric MTT assay was used to evaluate the anticancer activity of osimertinib/olaparib alone, Yunzhi alone, or their combination. When Yunzhi alone produced a complete concentration-cell growth inhibition curve, the combination of osimertinib/olaparib and Yunzhi (in fixed equipotent molar ratio) was analyzed by the median-effect analysis method. The combination index (CI) was calculated according to established method. CI < 1, = 1, and > 1 indicates synergistic, additive and antagonistic combination effects, respectively. On the other hand, if Yunzhi alone did not significantly inhibit cancer cell proliferation (i.e., IC₅₀ was not achieved), the enhancement of osimertinib/olaparib-induced cell growth inhibition by Yunzhi was calculated. Apoptosis assay and Western blot analysis were conducted to evaluate the apoptotic anticancer effect and inhibition of key oncogenic signaling molecules after treatment with the drug combinations. Moreover, as Yunzhi is widely believed to produce its biological effects by modulating the body’s immune system. Co-culture experiments of NSCLC and Jurkat (T cell)/THP-1 (macrophage) were conducted to investigate the contribution of immune response in the circumvention of osimertinib resistance by Yunzhi.

Results: Yunzhi alone exhibited different extent of anticancer activity in the panel of NSCLC and AML cell lines tested. Importantly, Yunzhi was found to potentiate the anticancer effect of osimertinib more preferentially in osimertinib-resistant NSCLC cell lines (NCI-H820 and H1975-OsiR) harboring MET amplification. Synergistic anticancer effect was observed for osimertinib+Yunzhi combination in NCI-H820 cells, which was accompanied by enhanced apoptotic effect and remarkable inhibition of Erk/Akt signaling pathways. Simultaneous combination, osimertinib-preceding-Yunzhi, or the vice versa combination sequence appeared to produce similar beneficial anticancer effect, which were all better than the two individual drugs alone. On the other hand, osimertinib+Yunzhi combination did not show appreciable cytotoxicity in a normal bronchial epithelial cell line BEAS-2B. For AML, Yunzhi was also shown to produce synergistic anticancer effect more significantly in olaparib-resistant cell lines than the corresponding sensitive parental cell lines, apparently circumventing olaparib resistance. In the macrophage-cancer cell co-culture model, Yunzhi was found to promote the polarization of THP-1-derived macrophages into the classical M1 subtype (pro-inflammatory and anti-tumoral), as evidenced by the increased concentration of the pro-inflammatory cytokines TNF-a and IL-6. This was accompanied by more significant inhibition of cancer proliferation and activation of apoptosis of the co-cultured NCI-H820 cells. In the T cell-cancer cell co-culture system, Yunzhi was also shown to potentiate the Jurkat T cell-mediated anticancer effect in a concentration-dependent manner, which was associated with increased IFN-g secretion in the supernatant medium and increased apoptosis in the co-cultured NCI-H820 cells.

Conclusion: Yunzhi was shown to overcome osimeritinib or olaparib resistance in human NSCLC and leukemia cell lines, respectively. More significant circumvention of osimertinib resistance in NSCLC was achieved in resistant cells harboring Met amplification. Results from the cancer cell-macrophage/T -cell co-culture suggests an important role played by Yunzhi-mediated anticancer immunity to circumvent osimertinib resistance in NSCLC. Our findings advocate further in vivo validation of the circumvention of drug resistance by Yunzhi in immunocompetent tumor-bearing mice.

Acknowledgements: Funding support from the SKL Open Fund (SKLOF-X) provided by the Institute of Chinese Medicine, The Chinese University of Hong Kong is greatly acknowledged.