TM-233, a Novel Analog of 1'-Acetoxychavicol Acetate (ACA), Induces Cell Death in Human Myeloma Cells by Inhibiting Both JAK/STAT and NF-κB Pathways: A Possible New Agent for Overcoming Bortezomib-Resistance.
Authors: Sagawa Morihiko , Tomikawa Tatsuki , Nemoto Tomoe , Tabayashi Takayuki , Watanabe Reiko , Tokuhira Michihide , Ri Masaki , Hashimoto Yuichi , Iida Shinsuke , Kizaki Masahiro 
Published: October 2019

Abstract
Abstract 2939
Multiple myeloma (MM) remains incurable despite the use of conventional and high-dose chemotherapy with stem cell transplantation. However, novel agents such as thalidomide, lenalidomide, and bortezomib have already achieved promising clinical results. In particular, bortezomib, a proteasome inhibitor, is widely used in the treatment of MM and has remarkable clinical effects in both refractory/relapsed and newly diagnosed patients. However, acquired drug resistance and serious toxicities such as peripheral neuropathy are often triggered by bortezomib in the clinical settings. Therefore, novel targeted agents with less toxicity are necessary to overcome bortezomib-resistance and improve clinical outcomes in MM. We have previously reported that 1'-acetoxychavicol acetate (ACA), a component of a traditional Asian condiment obtained from the rhizomes of the commonly used ethno-medicinal plant Languas galanga, has an inhibitory effect on NF-κB, and induces cell death in MM cells both in vitro and in vivo (Cancer Res, 2005; 65: 4417). With the aim of discovering more potent NF-κB inhibitors, we subsequently developed several ACA analogs based on quantitative structure-activity relationship (QSAR) analysis. This showed that a novel benzhydrol-type analog of ACA (TM-233) exhibited greater inhibition of NF-κB activity in MM cells when compared with ACA. In the present study, we examined the effects of TM-233 on various MM cells, including those resistant to bortezomib and we investigated the molecular mechanism of TM-233-induced cell death in these cells.
TM-233 inhibited cell proliferation and induced cell death in various MM cell lines (U266, RPMI8226, and MM-1S cells) in a time (0–48 hours)- and dose (0–5 μM)- dependent manner. Similarly potent activity was observed against primary MM cells. Exposure of bone marrow mononuclear cells from 3 patients with MM to TM-233 revealed selective activity under the same conditions as for cell lines. Moreover, TM-233 did not inhibit colony formation in CD34-positive cells from human umbilical cord blood; therefore, TM-233 did not influence the normal blood cells. We next investigated the effects of TM-233 on the various signaling pathways including JAK/STAT and NF-κB in MM cells. TM-233 activated apoptosis-related caspase-3, -8, -9, and PARP, suggesting that cell death related to TM-233 is mediated by both mitochondrial- and Fas-dependent pathways. Furthermore, treatment with TM-233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti-apoptotic Mcl-1 protein, but not that of Bcl-2 and Bcl-xL proteins. TM-233 directly bound and activated the transcription of the Mcl-1 gene promoter, suggesting that TM-233-induced cell death in MM cells down-regulated Mcl-1 via modulation of the JAK/STAT pathway. In addition, the DNA-binding activity of NF-κB in MM cells was inhibited by the treatment of TM-233. Further, TM-233 rapidly decreased the nuclear expression of NF-κB, but increased the accumulation of cytosol NF-κB, suggesting that TM-233 inhibits the translocation of NF-κB from the cytosol to the nucleus. These results indicate that TM-233 induced cell death in MM cells occurs via both JAK/STAT and NF-κB pathways. We finally examined the effects of TM-233 on bortezomib-resistant MM cells. We have recently established the bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ (Leukemia 2010; 24: 1506). We found that these cells have a unique point mutation, G322A, in the gene encoding the proteasome b5 subunit, resulting in bortezomib-resistance mediated through prevention of the accumulation of unfolded proteins and fatal ER stress. TM-233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells in a time- and dose-dependent manner. Interestingly, the combination of TM-233 and bortezomib significantly induced cell death in these bortezomib-resistant MM cells via inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in MM cells by acting according to a different mechanism from that of bortezomib, possibly inhibition of the JAK/STAT pathway.
In conclusion, TM-233, a novel analog of ACA, might be a more potent NF-κB inhibitor than ACA and could overcome bortezomib-resistance in MM cells. Further studies investigating clinical applications are necessary and may yield interesting results.

Disclosures:
Iida: Janssen Pharmaceutical K.K.: Honoraria. Kizaki:Janssen Pharmaceutical K.K.: Honoraria.