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Introduction: Triple-negative breast cancer (TNBC) is an aggressive breast cancer with shorter overall survival compared to other breast cancer types. One of the six molecularly-classified TNBC subtypes is the luminal androgen receptor subtype (LAR), which overexpresses androgen receptor (AR) and is dependent on AR for its growth. About 10-20% of TNBCs belong to the LAR subtype. Competitive AR antagonists, enzalutamide and bicalutamide, were effective in preclinical models of LAR TNBC and in clinical trials. This led us to hypothesize that potent selective AR degraders (SARDs), due to their ability to inhibit and degrade the AR, could provide a novel therapeutic approach for the treatment of LAR subtype of TNBC. Description: Western blots, cell line proliferation assays, and gene expression analyses were performed to evaluate novel small molecule SARDs. LAR TNBC cell lines and patient-derived xenografts (PDX) were utilized for in vivo evaluation of the SARDs. Once tumors grew to ~100-300 mm3, mice were randomized and treated orally for four weeks with vehicle, SARD UT-34, SARD UT-105, enzalutamide, or bicalutamide. Tumor volumes were measured twice weekly and tumors were collected at sacrifice for further analyses. Summary: SARDs bind to the N-terminus of the AR and have been characterized in preclinical advanced prostate cancer models. In this study, the SARDs were evaluated in preclinical models of LAR TNBC. Western blot for AR in LAR MDA-MB-453 cells demonstrated degradation of the AR protein by SARDs at low micromolar concentrations. Gene expression studies showed a complete inhibition of androgen-induced AR target gene transcription by the SARDs. Androgen-induced proliferation of MDA-MB-453 cells was inhibited by SARDs. MDA-MB-453 cells implanted subcutaneously in NOD SCID Gamma female mice grew robustly to 100-300 mm3 in 15-20 days. Treatment of tumor-bearing animals with the SARDs completely inhibited or regressed the tumors. Conclusion: These results support the findings that AR is the driver of MDA-MB-453 cell and tumor growth. SARDs with their unique mechanism of action may provide a new therapeutic option to women affected by the LAR subtype of TNBC. Disclosure: This work was partially supported by Oncternal Therapeutics and by an NCI supplement award to R01 (CA229164S1 to author RN). The SARD program has been licensed to Oncternal Therapeutics, Inc. by the University of Tennessee Research Foundation. Author RN is a consultant to Oncternal Therapeutics. Citation Format: Sarah Asemota, Kirsten Young Young, Suriyan Ponnusamy, Thirumagal Thiyagarajan, Dong-Jin Hwang, Yali He, James B. Breitmeyer, Gunnar F. Kaufmann, Duane D. Miller, Ramesh Narayanan. Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1235.

Antimitotics that target tubulin are among the most useful chemotherapeutic drugs, but their clinical activity is often limited by the development of multidrug resistance. We recently discovered the novel small-molecule DJ101 as a potent and metabolically stable tubulin inhibitor that can circumvent the drug efflux pumps responsible for multidrug resistance of existing tubulin inhibitors. In this study, we determined the mechanism of action of this drug. The basis for its activity was illuminated by solving the crystal structure of DJ101 in complex with tubulin at a resolution of 2.8Å. Investigations of the potency of DJ101 in a panel of human metastatic melanoma cell lines harboring major clinically relevant mutations defined IC50 values of 7–10 nmol/L. In cells, DJ101 disrupted microtubule networks, suppressed anchorage-dependent melanoma colony formation, and impaired cancer cell migration. In melanoma-bearing mice, DJ101 administration inhibited tumor growth and reduced lung metastasis in the absence of observable toxicity. DJ101 also completely inhibited tumor growth in a paclitaxel-resistant xenograft mouse model of human prostate cancer (PC-3/TxR), where paclitaxel was minimally effective. Our findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation tubulin inhibitor for cancer therapy. Significance: These findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation antitubulin drug for cancer therapy. Cancer Res; 78(1); 265–77. ©2017 AACR.

Interfering with microtubule dynamics is a validated approach for anticancer treatment and by selectively targeting the colchicine binding site on tubulin, microtubule destabilizing agents can evade mechanisms of drug resistance that commonly develop with other antimitotic agents such as taxanes and vinca alkaloids. We have recently reported the discovery of a potent and metabolically stable tubulin inhibitor (DJ101) that specifically targets the colchicine binding site on the beta tubulin subunit, disrupts tubulin polymerization and effectively circumvents drug efflux pumps that decrease the efficacy of existing tubulin inhibitors. To further pre-clinically evaluate DJ101 as a novel tubulin inhibitor and confirm its mechanism of action, we first visually demonstrated the ability of DJ101 to disrupt microtubule dynamics and elucidated the changes in microtubule structure and cell morphology through immunofluorescence techniques. Furthermore, we solved the crystal structure of DJ101 in complex with tubulin to determine its molecular interactions with the protein. We then demonstrated the significant in vitro potency of DJ101 against a panel of metastatic melanoma cell lines harboring major clinically relevant mutations observed in tumors and also validated its cytotoxicity against a broader array of NCI-60 cell lines. Further analysis revealed that DJ101 effectively thwarted anchorage-dependent melanoma colony formation and drastically hindered cell mobility and motility of those cancer cells. We further ascertained that DJ101 shows negligible off-target effects for major physiologically important receptors and ion channels, suggesting a good safety profile. Encouraged by the preliminary results from our in vitro assessment, we evaluated the in vivo activity of DJ101 in two different mouse models. At a dose of 30 mg/kg via i.p. injections, DJ101 nearly completely suppressed tumor growth in a human A375 melanoma xenograft model. It also appreciably inhibited metastasis potential in a murine B16F10 lung metastasis model at the same dose. In conclusion, we have described a microtubule destabilizing agent that targets the colchicine binding site on tubulin, confirmed its mechanism action, determined its potency in a vast array of cancer cell lines, particularly metastatic melanoma, demonstrated its inhibitory effect on cellular proliferation, motility and migration, and evaluated its anticancer potential in terms of tumor growth inhibition and metastasis in two in vivo models. Our findings offer a compelling rationale to further develop and advance DJ101 as a tubulin inhibitor for cancer therapy. Citation Format: Kinsie Arnst, Dong-Jin Hwang, Duane D. Miller, Wei Li. Novel tubulin inhibitor DJ101 targets the colchicine binding site and suppresses melanoma growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3223. doi:10.1158/1538-7445.AM2017-3223