Duxin Sun Research Articles

Abstract 4519: Discovery of highly potent, selective and efficacious STAT3 PROTAC degraders capable of achieving complete tumor regression

Abstract STAT3 (signal transducer and activator of transcription 3) is a transcription factor and a promising therapeutic targets for cancer and other human diseases. We have previously reported the discovery of SD-36 and SD-91 as potent, selective and highly efficacious STAT3 degraders. In the present study, we report the discovery and extensive evaluation of highly potent, selective and efficacious new STAT3 degraders designed using novel cereblon ligands and novel STAT3 ligands. Our most potent STAT3 degraders are >10-times more potent than SD-36 in inducing STAT3 degradation in cells and demonstrates >100-fold degradation selectivity over other STAT members. They achieve nanomolar IC50 values in leukemia and lymphoma cancer cell lines with activated STAT3. A single intravenous dose of these STAT3 degraders reduces the STAT3 protein for >48 hrs in xenograft tumor and native tissues, without reducing another other STAT members. Weekly administration of these STAT3 degraders is capable of achieving complete tumor regression in xenograft tumor models without any signs of toxicity. Taken together, these STAT3 degraders are promising lead compounds for extensive evaluation for the treatment of the treatment of human cancers and other human diseases in which STAT3 plays a key role. Citation Format: Haibin Zhou, Dimin Wu, Longchuan Bai, Ranjan K. Acharyya, Hoda Metwally, Donna McEachern, Bo Wen, Duxin Sun, Shaomeng Wang. Discovery of highly potent, selective and efficacious STAT3 PROTAC degraders capable of achieving complete tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4519.

Abstract 4510: Potent and orally efficacious PROTAC degraders of estrogen receptor α (ERα)

Abstract The estrogen receptor α (ERα) plays a crucial role in hormonally driven breast cancer, making it a clinically validated oncology target. Over the past 30 years, SERMs, SERDs, and AIs have been the mainstay of endocrine therapy for ER+ breast cancer, but relapse and resistance remains a significant challenge for metastatic breast cancer, leading to cancer recurrences, metastasis, and mortality. We report the discovery and extensive evaluations of potent and orally efficacious ERα degraders using the PROTAC technology through extensive optimization of the cereblon ligands, the linker and ER ligands. Our lead compounds (UM-ERD-3111 and UM-ERD-4001) achieved sub-nanomlar DC50 values in ER degradation and demonstrate excellent oral bioavailability. Oral administration of these ER degraders effectively reduces the ER wild-type and ER mutated proteins in tumor tissues in mice. Both ERD-3111 and UM-ERD-4001 are capable of inducing tumor regression in vivo and are more efficacious than ARV-471, a PROTAC ER degrader currently in phase 3 clinical development. UM-ERD-3111 and UM-ERD-4001 are highly promising compounds for extensive evaluations as potential new therapies for the treatment of ER+ human breast cancer. Citation Format: Rohan K. Rej, Zhixiang Chen, Ranjan K. Acharyya, Dimin Wu, Biao Hu, Guozhang Xu, Rakesh Nagilla, Hoda Metwally, Yu Wang, Bo Wen, Duxin Sun, Longchuan Bai, Shaomeng Wang. Potent and orally efficacious PROTAC degraders of estrogen receptor α (ERα) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4510.

Abstract 6534: Overcoming B regulatory cell induced STING resistance by dual functional albumin nanomedicine to prolong survival of pancreatic cancer

Abstract Pancreatic cancer is the most lethal form of cancer with a 5-year survival at 11% and lacks treatment options. Immunotherapy, specifically anti-PD-1/PD-L1 antibody, proves ineffective for pancreatic cancer patients due to the immunosuppressive tumor microenvironment (TME). STING agonist has emerged as the most effective immune modulator to regulate immunosuppressive TME to enhance the efficacy of immunotherapy across many different cancer types. However, two challenges hinder the use of STING agonist in pancreatic cancer: (A) STING agonist strongly stimulates induction of B regulatory cells (Bregs) in pancreatic tumor and lymph node, leading to an intrinsic resistance to STING agonist and a severe compromised efficacy. (B) Most STING agonists are used by local intra-tumor injection in clinical trials, which only shrinks the local tumors without inhibiting distal tumors or metastasis, whereas it is also not feasible for intra-tumor injection for pancreatic cancer in clinical trials. In response, we developed a first-in-class dual functional drug (SH-273) to overcome STING resistance by eliminating Bregs in both tumors and lymph nodes for long-term efficacy in pancreatic cancer. SH-273 has dual function to stimulates STING function (EC50 100 nM) and inhibits PI3Kγ (IC50 7 nM) that eliminate Bregs to overcome STING resistance. Interestingly, SH-273 achieved dual function with an opposite mechanism in regulating IRF3 phosphorylation in myeloid cells (increase) vs. in Bregs (decrease). In addition, we developed an albumin nanoparticle of SH-273 (Nano-273) for systemic delivery to enhance drug targeting to tumor and lymph node to activate systemic anticancer immunity. 3D imaging results indicated that albumin nano formulation greatly enhanced drug lymphoid draining and pancreatic tumor penetrating. Nano-273, combined with anti-PD-1, achieved long-term median survival of 200 days in LSL-KrasG12D; LSL-Trp53R172H/+; Pdx1cre/+ (KPC) mice (a substantial 67% increase from 120 days without treatment). The 80-day survival extension is significant as KPC mice mimic human pancreatic cancers with Kras/P53 mutations that are unresponsive to other available therapies. Single cell RNA-sequencing and flow cytometry revealed that Nano-273, combined with anti-PD-1, reduces Bregs by 5 to 7-fold while increasing other B cell subtypes by 4-fold in both pancreatic tumors and lymph nodes, compared to control group or group treated with STING agonist alone. These findings suggest that Nano-273 overcomes STING resistance by eliminating Bregs and induced systemic immunity to achieve long-term anti-tumor efficacy in pancreatic cancer. Citation Format: Chengyi Li, Shuai Mao, Hongyi Zhao, Djibo Mahamadou, Zhongwei Liu, Hanning Wen, Miao He, Meilin Wang, Jinsong Tao, Bo Wen, Trang Hoang, Binyamin Jacobovitz, Fei Wen, Wei Gao, Duxin Sun. Overcoming B regulatory cell induced STING resistance by dual functional albumin nanomedicine to prolong survival of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6534.

Abstract 4475: Discovery of an oral tricyclic STING agonist with superior pharmacokinetic properties and potent in vivo efficacy

Abstract Stimulator of interferon genes (STING) plays a pivotal role in regulating immune microenvironment for cancer immunotherapy. However, the use of most STING agonists has been hindered by the necessity for intra-tumoral or intravenous administration, underscoring the challenge in developing an orally bioavailable STING agonist with favorable pharmacokinetic (PK) properties. In this study, we discovered oral STING agonists featuring a novel tricyclic benzo[4,5]thieno[2,3-c]pyrrole-1,3-dione scaffold, thus achieving both superior PK properties and potent in vivo efficacy. Our lead compound, ZSA-51, exhibited nanomolar cellular STING activation activity (EC50 = 100 nM) in THP1 cells, surpassing the reported oral STING agonist MSA-2 by 32-fold (EC50 = 3200 nM). Furthermore, ZSA-51 demonstrated superior PK properties with an oral bioavailability of 49%, and robust in vivo antitumor activity both in colon and pancreatic cancers model upon oral administration. Notably, ZSA-51 displayed preferential distribution to the lymph nodes and spleen, enhancing its systemic immune-stimulating effects. The specificity of ZSA-51 was further confirmed by a comparison with a negative control compound (ZSA-52NC2) that was unable to stimulate in vitro and in vivo STING activity. Molecular docking and MD simulation confirmed that the active form of ZSA-51 stably bound to STING dimer with validated hydrogen bond network. Taken together, our findings suggest ZSA-51 as a promising oral STING agonist characterized by superior PK properties and potent in vivo efficacy, holding potential for future development for cancer immunotherapy. Citation Format: Hong-Yi Zhao, Zhongwei Liu, Shuai Mao, Miao He, Meilin Wang, Bo Wen, Wei Gao, Duxin Sun. Discovery of an oral tricyclic STING agonist with superior pharmacokinetic properties and potent in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4475.

Abstract 6057: Evaluation of a highly potent and selective STAT3 degrader as a new class of immunotherapy

Abstract STAT3 (signal transducer and activator of transcription 3) is a transcription factor and a promising therapeutic target for cancer and other human diseases. In addition to its role in regulation of tumor cells, STAT3 also plays a key role in regulation of immunity and is a promising therapeutic target for the development of new immuno-oncology drugs. Our laboratory has previously reported the development of potent and highly selective STAT3 degraders, including SD-36 and SD-91. Extensive optimization of SD-36 and SD-91 yielded new, highly potent, selective and efficacious STAT3 degraders. In direct comparison, our most potent, new STAT3 degrader (compound 1) is >50-times more potent than SD-36 in inducing STAT3 degradation in cells and demonstrates >500-fold degradation selectivity over other STAT members. A single intravenous administration of compound 1 in mice is highly effective in inducing complete depletion of STAT3 protein in tissues for 48-96 h without reducing the levels of other STAT proteins. Compound 1 is very effective in inhibition of tumor growth not only in tumor models responsive to immune checkpoint blockade (ICB) but also in tumor models resistant to ICB. Furthermore, combination of compound 1 with PD-1/PD-L1 antibodies greatly enhances the antitumor activity as compared to SD-1218 or ICB. Of significance, compound 1 is well tolerated in mice without any signs of toxicity in mice at highly efficacious doses. Our mechanistic studies show that STAT3 depletion has a major effect in modulation of the immune cells in mice. Collectively, our data suggest that selective STAT3 degradation hold great promise for new cancer immunotherapy. Citation Format: Longchuan Bai, Haibin Zhou, Jiajia Zhou, Dimin Wu, Ranjan Kumar Acharyya, Hoda Metwally, Donna McEachern, Bo Wen, Duxin Sun, Weiping Zou, Shaomeng Wang. Evaluation of a highly potent and selective STAT3 degrader as a new class of immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6057.

Abstract 3881: Discovery of highly potent, selective and efficacious STAT3 PROTAC degraders capable of achieving long-lasting tumor regression

Abstract STAT3 (signal transducer and activator of transcription 3) is a transcription factor and a promising therapeutic targets for cancer and other human diseases. We have previously reported the discovery of SD-36 and SD-91 as potent, selective and highly efficacious STAT3 degraders. In the present study, we report the discovery and extensive evaluation of new, highly potent, selective and efficacious new STAT3 degraders. In direct comparison, these compounds are >50-times more potent than SD-36 in inducing STAT3 degradation in cells and demonstrates >500-fold degradation selectivity over other STAT members. Proteomic analysis showed that our best lead compound (UM-STAT3-1218) only reduces the levels of STAT3 protein in cells over other >6,700 proteins. (UM-STAT3-1218) achieves low nanomolar IC50 values in leukemia and lymphoma cancer cell lines with activated STAT3. A single intravenous dose of (UM-STAT3-1218) at 3 mg/kg reduces the STAT3 protein for >4 days in xenograft tumor and native tissues, without reducing another other STAT members. Impressively, a single dose of (UM-STAT3-1218) at 3 mg/kg achieves complete and long-lasting tumor regression in multiple xenograft tumor models without any signs of toxicity. Taken together, (UM-STAT3-1218) is a highly promising STAT3 degrader for the treatment of the treatment of human cancers and other human diseases in which STAT3 plays a key role. Citation Format: Haibin Zhou, Dimin Wu, Longchuan Bai, Ranjan K. Acharyya, Hoda Metwally, Donna McEachern, Bo Wen, Duxin Sun, Shaomeng Wang. Discovery of highly potent, selective and efficacious STAT3 PROTAC degraders capable of achieving long-lasting tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3881.

Abstract 6735: A systemically administered non-nucleotide STING agonist in albumin nanoformulation with potent antitumor activity and low toxicity

Abstract Background: Stimulator of interferon genes (STING), a dimeric transmembrane adapter protein, plays a pivotal role in regulating tumor immune microenvironment and has been explored as a therapeutic strategy against tumors. Most STING agonists were tested in clinical trials using intratumoral injection with limited systemic efficacy. Several systemically administered STING agonists have progressed to clinical trials, with their efficacy yet to be determined, while their systemic toxicity may limit their application. Our study aimed to develop a novel systemically administrated non-nucleotide STING agonist formulated within albumin nanoparticles, demonstrating potent antitumor activity and low systemic toxicity. Methods & Results: We synthesized a novel non-nucleotide small molecule STING dimer ZSA-51D. The acid form of ZSA-51D exhibited a high binding affinity (EC 50: 1.3 nM) to human STING. ZSA-51D demonstrated potent STING activation in THP1-BlueTM ISG cells, with an EC50 of 5.1 nM. To enable systemic delivery, ZSA-51D was encapsulated in albumin nanoparticles (nano ZSA-51D) with a particle size of 115 nm. Surprisingly, nano ZSA-51D exhibited a tenfold increase in STING activation (EC50 of 0.44 nM) compared to free ZSA-51D. Furthermore, nano ZSA-51D demonstrated sevenfold greater activation of bone marrow-derived dendritic cells (EC 50 of 3.5 nM), and eightfold enhanced repolarization of bone marrow-derived macrophages (EC 50 of 4.2 nM) from M2 to M1 compared to free ZSA-51D. The in-vivo anticancer efficacy was evaluated on MC-38 and KPC 6620 xenograft in C57BL/6 mice using intravenous administration of ZSA-51D (1 mg/kg) or nano ZSA-51D, in combination with PD-1 antibody (α-PD1, 100 μg) for 5 dose every 3 days. The results revealed that nano ZSA-51D exhibited superior anticancer effects compared to free ZSA-51D. Remarkedly, nano ZSA-51D, in combination with α-PD1, completely eradicated cancer in the MC-38 xenograft model. These cured mice completely rejected the rechallenge with MC38 cells 120 days after the initial tumor inoculation, indicating potent and long-term anticancer immune memory for anticancer activity. In addition, we performed the toxicity evaluation of nano ZSA-51D and free ZSA-51D at the same dose regime as the efficacy study. Nano ZSA-51D shows no hematological and liver toxicity. However, free ZSA-51D has a sever local inflammation after injections, whereas nano ZSA-51D has no local toxicity. Conclusion: In this study, we developed a novel systemically administered non-nucleotide sting agonist formulated within albumin nanoparticles. Nano ZSA-51, in combination with α-PD1, demonstrated superior anticancer efficacy and low toxicity, holding the potential for future clinical trials. The significantly enhanced STING activation and in vivo efficacy of nano ZSA-51D warrant further investigation into its underlying mechanisms. Citation Format: Jinsong Tao, Hongyi Zhao, Zhongwei Liu, Hanning Wen, Chengyi Li, Qiuxia Li, Miao He, Bo Wen, Wei Gao, Duxin Sun. A systemically administered non-nucleotide STING agonist in albumin nanoformulation with potent antitumor activity and low toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6735.

Abstract 4509: Discovery of potent and highly efficacious STAT3 PROTAC degraders capable of achieving long-lasting tumor regression

Abstract STAT3 (signal transducer and activator of transcription 3) is a transcription factor and a promising therapeutic targets for cancer and other human diseases. We have previously reported the discovery of SD-36 and SD-91 as potent, selective and highly efficacious STAT3 degraders. In the present study, we report the discovery of highly potent, selective and efficacious new STAT3 degraders. These STAT3 degraders were designed using our high-affinity STAT3 ligands and high-affinity VHL-1 ligands, with UM-STAT3-3100 being the best. In direct comparison, UM-STAT3-3100 is >10-times more potent than SD-36 and SD-91 in inducing STAT3 degradation in cells and are highly selective over other STAT members in vitro and in vivo. A single intravenous administration of UM-STAT3-3100 resulted in complete and long-lasting depletion of STAT3 protein for >48 hrs in tumor and native tissues. Weekly administration of UM-STAT3-3100 at 10 mg/kg achieved complete and long-lasting tumor regression in animal models of human cancer without any signs of toxicity. UM-STAT3-3100 is a promising compound for extensive evaluation for the treatment of human cancers and other human diseases in which STAT3 plays a key role. Citation Format: Ranjan K. Acharyya, Longchuan Bai, Haibin Zhao, Dimin Wu, Metwally Hoda, McEachern Donna, Wen Bo, Duxin Sun, Shaomeng Wang. Discovery of potent and highly efficacious STAT3 PROTAC degraders capable of achieving long-lasting tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4509.

Abstract 4504: Discovery of CBPD-409 and CBPD-268 as highly potent and orally efficacious CBP/p300 PROTAC degraders for the treatment of castration-resistant prostate cancer

Abstract Prostate cancer remains the leading cause of cancer-related death globally in men. While current AR-targeted therapies are effective for the treatment of prostate cancer and improve patient survival, resistance in patients typically develops within 18 months. CBP/p300 are AR transcriptional co-activators and are promising therapeutic targets for the treatment of castration-resistant prostate cancer (CRPC). We developed a series of highly potent, selective, and orally efficacious CBP/p300 PROTAC degraders, with CBPD-409 being the best compound. CBPD-409 induces robust CBP/p300 degradation with DC50 0.2-0.4 nM and displays strong anti-proliferative effects with IC50 1.2-2.0 nM in VCaP, LNCaP and 22Rv1 AR+ cell lines. It has a favorable oral pharmacokinetic profile and achieves 50% of oral bioavailability in mice. A single oral administration of CBPD-409 at 1 mg/kg achieves >95% depletion of CBP/p300 proteins in the VCaP tumor tissue at 3 h and 24 h time points. CBPD-409 exhibits strong and dose/schedule-dependent tumor growth inhibition and is more potent and efficacious than two CBP/p300 inhibitors CCS1477 and GNE-049 and the AR antagonist Enzalutamide. To further improve the degradation potency and the oral bioavailability in rats, we developed a new class of CBP/p300 degraders based on our optimized CRBN ligand TX-16. Our efforts led to the discovery of CBPD-268 as an exceptionally potent, effective, and orally efficacious degrader of CBP/p300 proteins. In the VCaP, LNCaP and 22Rv1 cell lines, CBPD-268 induces consistent and robust CBP/p300 degradation with DC50 of ≤0.03 nM and Dmax >95%, leading to potent cell growth inhibition. CBPD-268 has excellent oral bioavailability in both mice and rats and has a good ADME profile. Oral administration of CBPD-268 at 0.3-3 mg/kg resulted in profound and persistent depletion of CBP and p300 proteins in tumor tissues and achieved strong antitumor activity in the VCaP and 22Rv1 xenograft tumor models in mice, including tumor regression in the VCaP tumor model. CBPD-268 was well tolerated in mice and rats and displayed a therapeutic index of >10. Taken together, CBPD-409 and CBPD-268 are highly promising CBP/p300 degraders for further extensive evaluations for the treatment of CRPC and other types of human cancers. Citation Format: Zhixiang Chen, Mi Wang, Dimin Wu, Lijie Zhao, Tianfeng Xu, Hoda Metwally, Yu Wang, Donna McEachern, Wei Jiang, Longchuan Bai, Jie Luo, Meilin Wang, Ruiting Li, John Takyi-Williams, Lu Wang, Qiuxia Li, Bo Wen, Duxin Sun, Arul M. Chinnaiyan, Shaomeng Wang. Discovery of CBPD-409 and CBPD-268 as highly potent and orally efficacious CBP/p300 PROTAC degraders for the treatment of castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4504.

Abstract 4506: Discovery of potent, highly selective and orally efficacious SMARCA2 degraders

Abstract In human non-small cell lung cancer, melanoma and other types of human cancers, the mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) helicase SMARCA4 is frequently mutated, which leads to inactivation of its functions. SMARCA2, a close homologous protein of SMARCA4, is an attractive synthetic lethality target for human cancers with SMARCA4 deficiency. Herein, we report the discovery and biological evaluation of potent, highly selective, orally efficacious SMARCA2 PROTAC degraders exemplified by UM-SMD-8801. UM-SMD-8801 has DC50 <10 nM and Dmax >90% against SMARCA2 and >1,000-fold degradation selectivity over SMARCA4. Consistently, UM-SMD-8801 potently inhibits cell growth in SMARCA4 mutated cancer cell lines with low nanomolar IC50 values and shows >100-fold weaker activity in SMARCA4 wild-type cancer cell lines. UM-SMD-8801 has a good overall pharmacokinetic profile and an excellent oral bioavailability in mice. Oral administration of UM-SMD-8801 is highly effective in reducing SMARCA2 protein by >90% in tumor tissues in mice, while having minimal effect on SMARCA4 protein. UM-SMD-8801 represents a very promising SMARCA2 degrader for extensive evaluation as a potential new therapy for the treatment of SMARCA4-deficient human cancers. Citation Format: Lingying Leng, Lin Yang, Wenbin Tu, Rohan Rej, Srinivasa Rao Allu, Liyue Huang, Wei Jiang, Yu Wang, Jeanne Stuckey, Farzad R Sarkari, Meilin Wang, Lu Wang, Bo Wen, Duxin Sun, Shaomeng Wang. Discovery of potent, highly selective and orally efficacious SMARCA2 degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4506.

Abstract 500: Discovering epigenetic regulators of cells states in triple negative breast cancers

Abstract The implementation of targeted therapies have been instrumental in achieving clinical responses for ER, PR, and HER2 receptor expressing breast cancers. However, triple negative breast cancers in themselves do not respond to such therapies and are highly heterogeneous diseases with significantly worse progression and survival. Our preliminary data and literature have also shown that in TNBC different cancer cell subpopulations are present in any given tumor, each with its own unique chemo-response and resistance. To discover novel targets regulating cancer cell states in TNBCs, we have created a reporter cell line using CRISPR/Cas9 mediated knock-in of fluorescent reporter proteins after the endogenous loci of major cell type markers in SUM149. Using this reporter line allows for the direct visualization of ALDH1A3+/- and CD24+/- cells, facilities tracking of distinct cell state transitions in a given cell, and allows for lineage tracing following cell divisions. These reporter cells were subjected to HTS-FACS analysis after exposure to a siRNA library of known epigenetic regulators. Following secondary screening, three siRNA targets are able to uniquely inhibit distinct subpopulations of cells within the cell line DCAF1 (ALDH-/CD24- cells), MLL2 (ALDH-/CD24+ cells), and TRIM24 (ALDH+ cells). In combination, these siRNA's proportionally eliminated all cell types. Due to the inherent heterogeneity of TNBCs we then sought to determine if efficacy was consistent or unique across different cell lines with varying percentages of CD44, CD24, and Aldefluor+ cell populations. Most notably, in MDA-MB-468 which contains similar cell population percentages to SUM149 each siRNA was capable of reducing cell proliferation. Similarly, TRIM24 was also capable of reducing cellular proliferation in BT20, MLL2 in MDA-MB-231, and DCAF1 in HCC1937. Knockdown of TRIM24 consistently reduced Aldefluor+ cells in MDA-MB-468 and BT20 cells. Finally, coupled with sc-Omics technologies we utilized this efficacy data identify a subset of TNBC patients likely to respond to therapies which will target these genes in the future. Citation Format: Joseph Patrick Burnett, Garrett Johnson, Nathan Truchan, Michael Brooks, Max S. Wicha, Duxin Sun. Discovering epigenetic regulators of cells states in triple negative breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 500.

Abstract 5727: Metabolic status and adaptability of breast cancer stem cells

Abstract Breast cancer stem cells (BCSCs) represent the subpopulation of malignant cells that cause tumor initiation, metastasis, and recurrence. BCSCs resist therapy with radiation and standard drugs, emphasizing the need to identify new vulnerabilities as therapeutic targets. Here, we investigated the metabolism of BCSCs at single-cell resolution using molecular imaging and scRNA sequencing techniques. We imaged retention of the fluorescent dye PKH26 or expression of a CRISPR/Cas9-engineered ALDH1A3-mCherry promoter-reporter to identify BCSCs and metabolic state of cells by two-photon microscopy with fluorescence lifetime imaging (FLIM) of endogenous NADH. We previously reported that sorted BCSCs exhibited enhanced metabolic plasticity relative to bulk tumor cells in 2D culture and rapidly metabolically adapted to the glycolysis inhibitor 2-deoxyglucose (2DG). Since cells rewire signaling and metabolism in 3D environments, we utilized two-photon microscopy to quantify metabolism in secondary mammospheres and living animals. In both intact spheres and orthotopic tumor xenografts, BCSCs marked by either fluorescent reporter utilized glycolysis to a greater extent than bulk cancer cells. We validated our findings by correlating cellular entropy as a marker for cancer stem cells with metabolic pathway preferences on a single-cell basis. When treated with 2DG, mammospheres showed increased OXPHOS and a significant decrease in the percentage of ALDH1A3-mCherry+ cells. These data 1) highlight capabilities of FLIM to measure the metabolism of single cancer cells in physiologic environments; 2) correlate molecular phenotypes with real-time molecular imaging profiles of metabolism and stem-ness; and 3) reveal that BCSCs rely on glycolysis, suggesting a potential target for metabolic therapy. Citation Format: Johanna M. Buschhaus, Ayse J. Muniz, Ming Luo, Joseph P. Burnett, Nathan A. Truchan, Michael D. Brooks, Brock A. Humphries, Kathy E. Luker, Joerg Lahann, Duxin Sun, Max S. Wicha, Gary D. Luker. Metabolic status and adaptability of breast cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5727.

Abstract A08: Resolving STING-mediated tumor immune microenvironment shift at single-cell resolution

Abstract Background: The detection of double-stranded cytosolic DNA by cyclic GMP-AMP synthase (cGAS) leads to activation of stimulator of interferon genes (STING) and to the production of type-I interferon (IFN-I) as part of host defense mechanism. Recently, 2’3’ cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), an endogenous activator of STING, has been shown to be a promising antitumor agent through the activation of innate and adaptive immunity. However, the mechanism by which cGAMP results in a tumor-specific T-cell expansion in the tumor microenvironment (TME) remains unclear. In addition, how highly anionic therapeutic STING agonists penetrate the hydrophobic phospholipid bilayer membrane to induce immune activation remains unknown. Materials and Methods: We propose that serum albumin is an endogenous carrier of cGAMP into the cell cytoplasm. The efficacy of albumin-cGAMP on IFN-I signaling was evaluated using real-time PCR in human monocytic cell line THP-1, as well as primary mouse bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMDMs). To examine the effects of albumin-cGAMP on macrophage populations, flow cytometry was performed on BMDMs. Efficacy of albumin-cGAMP was examined in vivo using syngeneic mouse model of oral cancer, followed by a 40-marker panel for mass cytometry (CyTOF) to characterize the immune landscape at single-cell resolution. Results and Discussion: We observed that the albumin-cGAMP mixture potently enhances the IFN-I signatures in THP-1 cells, BMDCs and BMDMs. Analysis of BMDMs reveals that cGAMP alone has a modest effect on macrophages and that albumin significantly increases cGAMP-induced M1/M2 polarization. Data analysis of CyTOF using SPADE and CITRUS reveals that the activation of STING results in significant reduction in subsets that compromise antitumor responses, such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs). An unbiased viSNE analysis reduces the high-dimension profiling data onto two dimensions and shows that CD4, CD8, B220, CD206, MHC-II, Dx5, F4/80, and Ly-6G are the main differentiating markers upon STING induction. Conclusion: Taken together, this work presents albumin as a potential physiologic carrier of STING agonist cGAMP, and we characterize STING-mediated immune landscape change in the TME at single-cell resolution. Citation Format: Yee Sun Tan, Blake R. Heath, Brett D. Hill, Xiaobo Luo, Syed M. Rizvi, Toktam Moghbeli, Hongxiang Hu, Erica Siismets, Haitao Wen, Yuying Xie, Duxin Sun, Simon Young, Weiping Zou, Fei Wen, Yu L. Lei. Resolving STING-mediated tumor immune microenvironment shift at single-cell resolution [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A08.

Abstract 968: Targeting intratumoral heterogeneity in TNBC with novel combinations of FDA-approved drugs

Abstract Triple negative breast cancer, like so many other solid tumors, has intratumoral heterogeneity which makes it difficult to treat. It is paramount to understand the question, “where does the intratumoral heterogeneity come from?” To that end, we developed a dual color reporter cell line using CRISPR/Cas9 to precisely knock in fluorescent proteins, mCherry and EGFP, behind CSC marker ALDH1A3 and differentiation marker CD24 respectively, in the TNBC cell line SUM149. With fluorescent microscopy we are able to track individual cells over time, view symmetrical and asymmetrical self-renewal, differentiation, and lineage restriction. Through direct observation we determined that the intratumoral heterogeneity comes from CSCs. In short, CSCs give rise to all the different cell types found within TNBC. Once we established the reporter cell line we wanted to ensure that any response to therapeutics was the same as in the parental SUM149 cell line. In the past, we have treated parental SUM149 with docetaxel or sulforaphane. We therefore treated our reporter cells with either docetaxel or sulforaphane. The sulforaphane treatment resulted in a significant reduction of CSCs while the docetaxel resulted in significantly more CSCs, both results matched the results from parental SUM149. To our surprise the docetaxel treatment result was due to the significant de-differentiation of mCherrylow to mCherryhigh cells. The de-differentiation and incomplete elimination of the heterogeneous cell populations pose a significant problem for TNBC patients, one that must be addressed. In order to overcome this issue, we will use our reporter cells to test already approved FDA drugs to find and evaluate novel combinations for their ability to completely eliminate the heterogeneous cell populations found within TNBC. Citation Format: Nathan A. Truchan, Joseph P. Burnett, Duxin Sun. Targeting intratumoral heterogeneity in TNBC with novel combinations of FDA-approved drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 968.

Abstract 3220: Enhancing breast cancer immunotherapy via IONP-mediated photothermal therapy

Abstract Despite immunotherapy by harnessing host’s own immune system to fight against cancer has elicited promising durable responses in metastatic melanoma and lung cancer patients, its efficacy in treating breast cancer remains to be low. In this work, we demonstrate that sequential photothermal therapy (PTT), mediated with systemically administered stealthy iron oxide nanoparticles (IONPs), can significantly enhance checkpoint blockade-based immunotherapy for breast cancer. Our data suggests that the combination therapy when combining IONP-mediated PTT with anti-CTLA-4 therapy results in a synergistic antitumor effect to completely inhibit established 4T1 murine breast tumor growth in BALB/c mice, while control treatments, including the antibody alone or IONP-mediated PTT alone, fail to do so. Our data further reveal that the enhanced antitumor effect may be attributed by the effective regulation of the suppressive tumor immunity by IONP-mediated PTT. Our flow cytometry data suggests that CD4+FoxP3+ regulatory T-cells (Tregs) are effectively depleted via IONP-mediated PTT, with a significant reduction of the Treg percentage from 42.2 ± 15.8% (control) to 11.9 ± 6.5% (p < 0.05). Our data further suggest that IONP-mediated PTT significantly reduces suppressor-cell-attractive cytokine granulocyte-colony stimulating factor (G-CSF) secretion, which is the driving force for Treg recruitment. Upon effective regulation of suppressive tumor immunity, the proportion of CD8+ T-cells in tumor tissues is significantly increased from 5.6 ± 2.1% (control) to 37.5 ± 6.9% (p < 0.001) following IONP-mediated PTT. Interestingly, our data suggest that Treg elimination and CD8+ T-cell activation can only be achieved through sequential PTT, not a single dose of PTT, suggesting that the suppressive contribution from the newly responding immune cells following initial antitumor treatment, and the sequential PTT may provide an effective way to not only deplete primary resident suppressive cells, but also newly responding ones. Our data further suggests that the combination therapy against primary tumors elicits a systemic immune rejection of cancer cells at distal sites, while this dramatic immune response is lost when CD8+ T-cells are depleted using anti-CD8 antibody, suggesting the immune rejection triggered by IONP-mediated PTT is through CD8+ T-cell-mediated immune responses. Our data also suggests that the majority of the combination treatment-cured mice can reject re-challenged 4T1 cancer cells, indicating memory T-cell immune surveillance. In conclusion, this study may provide an effective and translational nanomedicine-based strategy to effectively regulate suppressive tumor immunity and turn “cold” breast tumors into “hot”, thereby to significantly enhance checkpoint blockade-based breast cancer immunotherapy. Citation Format: Hongwei Chen, Xin Luan, Hayley Paholak, Joseph Burnett, Nicholas Stevers, Kanokwan Sansanaphongpricha, Miao He, Alfred Chang, Qiao Li, Duxin Sun. Enhancing breast cancer immunotherapy via IONP-mediated photothermal therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3220.

Abstract 1970: Novel Mcl-1 inhibitor effectively restores in vivo regorafenib sensitivity in resistant colorectal cancer

Abstract Colorectal cancer (CRC) is one of deadliest malignancies and approximately 25% of patients present with metastatic disease at the time of diagnosis, necessitating efficacious therapeutic options. Treatment of CRC has been greatly improved by targeted therapies such as the FDA-approved multi-kinase inhibitor, regorafenib, which elicits its anti-cancer effects by inducing apoptosis. Ours and other studies identified the anti-apoptotic Mcl-1 protein as a significant resistance factor to regorafenib treatment in CRC, associated with inactivating mutations in the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7). These mutations result in abrogated Mcl-1 degradation, which contributes to survival of cancer cells and ineffective regorafenib induced apoptosis. Therefore, there is sound rationale for Mcl-1 inhibition to circumvent FBW7 inactivity and restore the efficacy of regorafenib. Our group has discovered and developed several classes of selective small molecule Mcl-1 inhibitors that bind in the conserved BH3 groove. In HCT116 colon cancer cells, the lead compound, 483LM, engages endogenous Mcl-1 as demonstrated by a CETSA assay and leads to a rapid accumulation of Mcl-1 protein levels, indicative of protein stabilization. 483LM induces BAX/BAK dependent apoptosis in cancer cells, upregulates the pro-apoptotic protein, Noxa, and displays favorable in vivo DMPK properties. Using a genetic Mcl-1 knock-in (KI) CRC cell line to mimic the effect of FBW7 loss, we confirmed the in vitro resistance of these KI cells to single agent regorafenib treatment and restored apoptosis when treated in combination with 483LM. Importantly, the in vivo combination treatment of 483LM and regorafenib completely overcome Mcl-1-mediated regorafenib resistance in HCT116 KI CRC xenograft tumors. Pharmacodynamic evaluation of tumor samples revealed significant caspase-3 activation in 483LM treated tumors. The proximity ligation assay revealed the ability of 483LM to induce significant disruption of PUMA:Mcl-1 complexes in treated tumor samples. These data confirm the previous reports of the involvement of Mcl-1 in regorafenib resistance in CRC and provides rationale for further investigation into the clinical application of pharmacological Mcl-1 inhibition in combination therapies to combat resistance. In addition, these results provide evaluation and characterization of a novel Mcl-1 selective small molecule inhibitor with potent in vivo activity and potential to treat various malignancies. Citation Format: Karson J. Kump, Jingshan Tong, Ahmed S. Mady, Lei Miao, Siwei Li, Duxin Sun, Jian Yu, Lin Zhang, Zaneta Nikolovska-Coleska. Novel Mcl-1 inhibitor effectively restores in vivo regorafenib sensitivity in resistant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1970.

Abstract 2007: A novel CRISPR/Cas9 reporter system to monitor TNBC cancer stem cells in real time

Abstract Increasing evidence suggests that breast cancers arise from so-called cancer stem cells (CSC), which, consistent with their normal adult counterpart, are uniquely capable of long term self-renewal and differentiation into all bulk cancer cell types that exhibit unique epigenetic states within a tumor. While FACS has been instrumental to isolating these cells and quantifying efficacy of CSC targeted drugs in vitro and in vivo, having to remove cells from their native environment is an inherent limitation, thereby only providing a snapshot of information with each experiment. In order to directly visualize CSC dynamics in real time the CRISPR/Cas9 system was used to precisely knockin mCherry directly downstream of the final exon of ALDH1A3 in SUM149 triple-negative breast cancer cells. With regard to CSC properties, ALDH1A3 high cells were shown to be dramatically correlated with the ALDEFLUOR assay and functionally, increased ammosphere and tumor formation in vitro and in vivo. Strikingly, a single ALDH1A3 high cell was capable of reconstituting the entire heterogeneity of the parental cell line. With the ability to directly visualize CSCs we sought to utilize live cell imaging to observe population changes in response to varying therapeutics. As evident by mCherry fluorescence intensity we confirm previous findings that suggest conventional chemotherapeutics such as docetaxel enrich for CSCs while the natural product sulforpahane reduces the population. Taken together, we believe our mCherry reporter cells could be adapted to rapidly screen for novel inhibitors of CSCs in triple-negative breast cancers. Citation Format: Nathan A. Truchan, Johanna Buschhaus, Michael Brooks, Andre Halabu, Hongwei Guo, Hebao Yuan, Kathryn Luker, Max Wicha, Duxin Sun, Joseph P. Burnett. A novel CRISPR/Cas9 reporter system to monitor TNBC cancer stem cells in real time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2007.

Abstract 4604: Long noncoding RNA Linc00162, as a novel prognostic marker, modulates cancer stem cell properties in triple-negative breast cancer

Abstract Trastuzumab is an effective treatment for HER2+ breast cancer. However, trastuzumab resistance in HER2+ breast cancer may induce the epithelial-to-mesenchymal transition (EMT), transforming HER2+ to triple-negative breast cancer with cancer stem cell properties in some cancer cell populations. Long noncoding RNAs (lncRNAs) have been reported to be upregulated in many types of cancer tissues, and might play significant regulatory roles in cancer progression. However, the mechanisms of lncRNA in trastuzumab resistance still remain largely unknown. Here, we systematically analyzed the levels of LincRNA between BT474 (HER2+trastuzumab-sensitive) and the PTEN-deficient trastuzumab-resistant derivative (BT474-PTEN-LTT) by using RNA-sequencing, and identified that Linc00162 was significantly overexpressed in BT474 PTEN- LTT cells. In vitro functional assay indicated that downregulation of Linc00162 in BT474 PTEN-LTT cells completely inhibited the cell proliferation. To further evaluate the role of Linc00162 in BCSC self-renewal, we found that inhibition of Linc00162 led to a significant reduction in the number and the size of LTT cells sphere by using the mammosphere formation assay. Next, we examined Linc00162 expression levels in breast cancer tissues, and identified that Linc00162 was markedly increased. Clinicopathologic relevance analysis showed that Linc00162 expression was correlated with advanced clinical stage, lymph node metastasis and the depth of invasion. Furthermore, upregulation of Linc00162 was correlated with TRNB phenotype. In Kaplan-Meier survival analysis, high levels of Linc00162 was significantly associated with unfavorable survival in breast cancer patients. These findings suggest that Linc00162 can be used as a clinically relevant novel biomarker in breast cancer and may serve as a target for future drug development. Citation Format: Lichao Sun, ChangChing Lin, Joseph Burnett, Hebao Yuan, Duxin Sun. Long noncoding RNA Linc00162, as a novel prognostic marker, modulates cancer stem cell properties in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4604.

Abstract LB-053: Tracing redox-regulated breast cancer stem cell state transition using a dual fluorescent reporter system

Abstract Breast cancer stem cells (BCSCs) display plasticity allowing them to transition between quiescent mesenchymal- (M) and proliferative epithelial-like (E) states. This dynamic equilibrium of BCSCs in distinct M- and E-like states coordinately drives tumorigenesis, metastasis and treatment resistance. Despite the functional significance of CSC plasticity in tumor development and progression, tracing of BCSC state transitions under physiologic conditions is hampered by the lack of a functional dual fluorescent reporter to monitor state dynamics of BCSCs at single cell resolution. To this end, we developed a dual fluorescent reporter to assess EMT-MET state transitions of BCSCs in real-time. As E-BCSCs express high levels of ALDH1A3, we generated an ALDH1A3 fluorescent reporter system utilizing Crisper-Cas 9 mediated knockin of mCherry downstream of the ALDH1A3 promotor. To trace M-BCSCs that express high levels of CD44 and vimentin, we constructed a lentiviral reporter expressing GFP driven by a portion of the vimentin promotor that is specifically expressed in mesenchymal CD44+ cells. Dual reporter cells were generated by infection of ALDH1A3_mCherry expressing cells with the lentivirus expressing Vimentin_GFP. To assess stem cell characteristics of these reporter cells, we first examined their sphere-forming capacity. Both GFP-mCherry+ (mCherry+) and GFP+mCherry- (GFP+) cells have significantly higher capacity to form tumor spheres than GFP-mCherry- (DN) cells while the GFP+mCherry+ (DP) cells harbor the highest sphere-forming ability. To determine the relationship between cells expressing these reporters, we generated tumor spheres from single cells and examined the fluorescent pattern of resultant tumor spheres. While spheres generated from DP cells display mixed phenotypes of cells expressing mCherry and/or GFP, spheres derived from mCherry+ cells maintain mCherry+. Interestingly, spheres derived from GFP+ cells generated three distinct phenotypes with some spheres expressing strong GFP and weak mCherry fluorescence while others expressing strong mCherry but weak or no GFP signal. These distinct phenotypes of tumor spheres derived from GFP+ cells suggest that M-BCSCs possess capacity to transition to a dual (GFP+mCherry+) or epithelial-like (GFP-mCherry+) state in order to form tumor spheres at non-attachment culture conditions that are normally associated with increased oxidative stress. These results suggest that the DP cells are the most primitive, with the greatest tumor sphere forming capacity and multistate differentiation potential followed by the Vimentin_GFP+ cells, ALDH1A3_mcherry+ cells and DN cells. By time-lapse microscopy, we further monitored how metabolic/oxidative stress induced by glycolytic inhibitor 2DG or H2O2 affects BCSC state dynamics at single cell resolution. We found that oxidative stress promotes the transition of GFP+mCherry- M-BCSCs to GFP+mCherry+ (bipotent) and GFP-mCherry+ (E-like) BCSCs. In contrast, the antioxidant N-acetyl cysteine promoted the transition of bipotent and E-like BCSCs to a GFP+mCherry- M-like state. These studies demonstrate that the redox state of the tumor microenvironment regulates state transitions of BCSCs. This dual fluorescent reporter system should prove useful in monitoring CSC state transitions under more physiologically relevant in vivo conditions. Citation Format: Ming Luo, Michael Brooks, Joseph Burnett, Nathan Truchan, Duxin Sun, Max S. Wicha. Tracing redox-regulated breast cancer stem cell state transition using a dual fluorescent reporter system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-053.

Abstract 2004: Identification of MLK4 as a novel regulator of cancer stem cells in triple-negative breast cancer

Abstract Cancer stem cells (CSCs) are a small population of cancer cells that are capable of self-renewal and tumor initiation. As a result, targeting CSCs is a potential therapeutic strategy for preventing cancer relapse and metastasis. Mixed lineage kinase 4 (MLK4), a serine/threonine kinase, is known to regulate mesenchymal glioma stem cells and to drive tumorigenesis in colorectal cancer. We found that MLK4 was highly expressed in triple-negative breast cancer (TNBC) compared to other breast cancer subtypes according to the database from The Cancer Genome Atlas. Furthermore, multiple datasets indicated that higher expression of MLK4 was associated with poor prognosis in breast cancer. Despite the correlation of MLK4 and clinical outcome, the function of MLK4 in breast cancer is still not known. In this study, we found that knockdown of MLK4 significantly decreased secondary mammosphere formation, the CD44+/CD24- CSC population, invasion and migration in TNBC cell lines. In addition to suppressing CSC phenotypes in vitro, knockdown of MLK4 also inhibited tumor growth in NOD/SCID mice. Secondary transplantation of tumor cells demonstrated that silencing of MLK4 significantly decreased tumor-initiating cell frequency. As accessed by qPCR, knockdown of MLK4 led to decreased expression of mesenchymal genes (CD44 and VIM) and concomitant increased expression of epithelial genes (CDH1 and claudin genes). Immunohistochemical staining revealed that knockdown of MLK4 in xenografts resulted in increased expression of CK19, a luminal-epithelial marker. These studies indicate that MLK4 plays an important role in the function of mesenchymal-like CSCs in TNBC. Silencing of MLK4 in TNBC cell lines can transform CSC in these tumors to a more differentiated epithelial cell state. Together, these studies identify that MLK4 is a potential therapeutic target for CSCs in TNBC. Citation Format: Chang-Ching Lin, Miao-Chia Lo, Rebecca Moody, Nicholas Stevers, Samantha Tinsley, Mari Gasparyan, Max Wicha, Duxin Sun. Identification of MLK4 as a novel regulator of cancer stem cells in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2004.