Qin, Bo

Abstract Zinc finger E-box–binding homeobox 1 (ZEB1) is a transcription factor that can promote tumor invasion and metastasis by inducing epithelial-to-mesenchymal transition (EMT). To date, regulation of ZEB1 by RAS/RAF signaling remains unclear, and few studies have examined posttranslation modification of ZEB1, including its ubiquitination. In human colorectal cancer cell lines with RAS/RAF/MEK/ERK activation, an interaction of ZEB1 with the deubiquitinase ubiquitin-specific protease 10 (USP10) was identified whereby USP10 modifies ZEB1 ubiquitination and promotes its proteasomal degradation. Regulation of the USP10–ZEB1 interaction by MEK–ERK signaling was shown whereby constitutive activation of ERK can phosphorylate USP10 at Ser236 to impair its interaction with ZEB1 and enable ZEB1 protein stabilization. Stabilized ZEB1 was shown to promote colorectal cancer metastatic colonization in a mouse tail vein injection model. Conversely, MEK–ERK inhibition blocked USP10 phosphorylation and enhanced the USP10–ZEB1 interaction shown to suppress ZEB1-mediated tumor cell migration and metastasis. In conclusion, we demonstrate a novel function of USP10 in the regulation of ZEB1 protein stability and its ability to mediate tumor metastasis in a preclinical model. Implications: The MEK–ERK-regulated interaction of USP10 with ZEB1 can promote the proteasomal degradation of ZEB1 and thereby suppress its demonstrated ability to mediate tumor metastasis.

Abstract Androgen receptor (AR) is expressed in 80% to 90% of estrogen receptor α–positive (ER+) breast cancers. Accumulated evidence has shown that AR is a tumor suppressor and that its expression is associated with improved prognosis in ER+ breast cancer. However, both a selective AR agonist (RAD140) and an AR inhibitor (enzalutamide, ENZ) have shown a therapeutic effect on ER+ breast cancer, so the potential for clinical application of AR-targeting therapy for ER+ breast cancer is still in dispute. In this study, we evaluated the efficacy of ENZ and RAD140 in vivo and in vitro in AR+/ER+ breast cancer models, characterizing the relationship of AR and ER levels to response to AR-targeting drugs and investigating the alterations of global gene expression and chromatin binding of AR and ERα after ENZ treatment. In the AR-low setting, ENZ directly functioned as an ERα antagonist. Cell growth inhibition by ENZ in breast cancer with low AR expression was independent of AR and instead dependent on ER. In AR-high breast cancer models, AR repressed ERα signaling and ENZ promoted ERα signaling by antagonizing AR. In contrast, RAD140 activated AR signaling and suppressed AR-high tumor growth by deregulating ERα expression and blocking ERα function. Overall, analysis of the dynamic efficacies and outcomes of AR agonist, and antagonist in the presence of different AR and ERα levels reveals regulators of response and supports the clinical investigation of ENZ in selected ER+ tumors with a low AR/ER ratio and AR agonists in tumors with a high AR/ER ratio. Significance: The ratio of androgen receptor to estrogen receptor in breast cancer dictates the response to AR-targeted therapies, providing guidelines for developing AR-directed treatment strategies for patients with breast cancer.

Abstract Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor regulating epithelial to mesenchymal transition (EMT). To date, regulation of ZEB1 by key signaling pathways including RAS/RAF/MEK/ERK remains unclear, and few studies have focused on molecular associations or functional alterations of ZEB1 by post-translational modification. Here, we report an interaction of ZEB1 with the deubiquitinase USP10 that is regulated by MEK-ERK signaling. USP10 removes the K27 linkage ubiquitination chain on ZEB1 that enhances K48 polyubiquitination and promotes ZEB1 proteasomal degradation. However, constitutive activation of ERK can phosphorylate USP10 at Ser236 to impair its interaction with ZEB1 and, thereby, stabilize ZEB1 protein shown to promote colorectal cancer metastasis in mice. Conversely, inhibition of MEK-ERK blocks USP10 phosphorylation to enhance the USP10-ZEB1 interaction which can suppress ZEB1-mediated tumor cell migration and metastasis in vivo. Together, these data demonstrate a novel function of USP10 in the regulation of ZEB1 ubiquitination and protein stability that can inhibit ZEB1-mediated tumor metastasis.

Approximately one‐third of patients with metastatic castration‐resistant prostate cancer (CRPC) exhibited primary abiraterone resistance. To identify alternative treatment for abiraterone nonresponders, we performed drug discovery analyses using the L1000 database using differentially expressed genes identified in tumor biopsies and patient‐derived xenograft (PDX) tumors between abiraterone responders and nonresponders enrolled in PROMOTE trial. This approach identified 3 drugs, including topoisomerase II (TOP2) inhibitor mitoxantrone, CDK4/6 inhibitor palbociclib, and pan‐CDK inhibitor PHA‐793887. These drugs significantly suppressed the growth of abiraterone‐resistant cell lines and PDX models. Moreover, we identified 11 genes targeted by all 3 drugs that were associated with worse outcomes in both the PROMOTE and Stand Up To Cancer cohorts. This 11‐gene panel might also function as biomarkers to select the 3 alternative therapies for this subgroup of patients with CRPC, warranting further clinical investigation.

Abstract Background: Hormone receptor-positive (HR+) breast cancer (BC) accounts for ~60-70% of all BC cases. CDK4/6i combined with endocrine therapy (ET) is the standard of care for first- and second-line therapy of patients with HR+ metastatic breast cancer (MBC) based on substantial improvements in progression free survival (PFS) and overall survival compared to ET alone. Unfortunately, most patients (pts) develop resistance to CDK4/6i. The mechanisms underlying CDK4/6i resistance have not been fully elucidated. Robust in vivo CDK4/6i resistant animal models from pts with de novo and acquired CDK 4/6i resistance are lacking. Patient-derived xenografts (PDXs) are important preclinical tools in oncology to advance our understanding of cancer biology and to evaluate drug response phenotypes. Unfortunately, the establishment of sustainable PDXs from pts with HR+ BC is much more difficult than from HR-negative BC. In order to obtain clinically relevant models that can be utilized to better understand mechanisms of primary and acquired resistance to ET and CDK4/6i, we report the development of PDXs from a prospective study in which percutaneous biopsies were obtained from women receiving palbociclib for the treatment of HR+ advanced or MBC (PROMISE; NCT03281902). Methods: PROMISE (Prospective Study to Evaluate the Role of Tumor Sequencing in Women Receiving Palbociclib for Advanced Hormone Receptor (HR)-Positive Breast Cancer) is a prospective trial which enrolled pts with HR+, HER2-negative MBC who planned to start treatment with palbociclib and letrozole (1st line [FL]) or palbociclib and fulvestrant (2nd line [SL]). For pts enrolled at Mayo Clinic Rochester (MCR), serial tumor biopsies were obtained prior to the start of treatment and at disease progression to establish PDXs. Because baseline take rate was low, we amended the protocol to attempt PDX generation after the 2nd cycle (C2) of therapy. Tissues were received within an hour of excision and were orthotopically implanted in the mammary fat pad of 6~8-week-old nonobese diabetic (NOD)/SCID/IL-2g-receptor null (NSG) mice. Tumor growth was defined as tumor volume ≥ 50 mm3. Tumor take rate was defined as percent of patients with development of at least one stably transplantable (passed at least for two generations) xenograft that was pathologically confirmed as BC. Results: Of the 50 pts enrolled at MCR, 37 were FL pts (2 ineligible) and 13 were SL pts (1 ineligible). Fresh tumor samples obtained pre-treatment from 43 eligible pts (32 FL), after C2 from 17 eligible pts (13 FL), and at disease progression from 10 eligible pts (5 FL) were implanted. Tumor growth was seen in at least one mouse from a pre-treatment specimen in 1 (3.1%) FL pt (confirmed BC) and 4 (36.4%) SL pts (2 confirmed BC); from end of C2 specimens in 2 (15.4%) FL pts and 2 (50%) SL pts (1 confirmed BC); and at progression in 1 (20%) FL pt and 2 (40%) SL pts (1 confirmed BC). The take rate from pre-treatment samples among FL pts was 1/32 (3.1%; 95% CI: 0.08-16.2%). Take rates in other groups are pending histological review. Discussion: In pts receiving palbociclib for HR+ MBC, the take rate using a pre-treatment percutaneous biopsy was low (3.1%) in the FL setting. However, higher growth rates were seen in the SL setting. The observation of higher growth rates at the end of C2 and at progression may relate to the activation of G1/S that occurs with withdrawal of palbociclib, and should be further studied. PDX remains a valuable tool for the evaluation of tumor biology and the development of new therapeutics, especially for those models established from pts with palbociclib and ET resistance. Proteogenomic and sequencing studies are ongoing to fully characterize these PDXs. Citation Format: Jia Yu, Vera J. Suman, Jun He, Jason P. Sinnwell, Ann M. Moyer, Bo Qin, Yayun Gu, Huan Zhang, Jodi M. Carter, Krishna R. Kalari, Brendan P. McMenomy, Minetta C. Liu, Tufia C. Haddad, Kathryn J. Ruddy, Fergus J. Couch, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard Weinshilboum, Ciara O'Sullivan, Matthew P. Goetz, Liewei Wang. Patient-derived xenografts (PDXs) generated from hormone receptor-positive breast cancer (BC) before and after cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) treatment: Initial findings from the PROMISE study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-07.

Abstract Background Colorectal cancers (CRCs) are intrinsically drug resistant tumors with frequent low or absent tumor cell PD-L1 expression. Previously, we reported that PD-L1 depletion in CRC cells can confer chemoresistance. Given that JNK signaling has been implicated in cell survival and drug resistance in CRC cells, we examined the role of JNK and its regulation in chemoresistance in CRC cells with loss of PD-L1. Materials & MethodsHuman CRC cell lines (RKO, DLD1 and HCT15) were utilized and parental and PD-L1 knockout/knockdown cells were generated. Cells were treated with a JNK1-3 inhibitor (JNK-IN-8) alone or combined with a MEK1/2 inhibitor (cobimetinib), CPT-11 or oxaliplatin. Protein/protein interaction was analyzed by immunoprecipitation (IP); protein/RNA interaction was analyzed by RNA IP assay. RNA level and half life were measured by QRT-PCR. Apoptosis was measured by cleaved caspase-3 and quantified by Annexin V labeling using FACS. Colony formation assay was performed. ResultsPD-L1 depletion in CRC cells was shown to enhance JNK activity that was due to reduced mRNA stability of the upstream CYLD deubiquitinase. Since PD-L1 competes with the ribonuclease EXOSC10 for binding to CYLD mRNA, loss of PD-L1 increases the interaction of EXOSC10 and CYLD mRNA resulting in its ribonuclease-mediated degradation and activation of JNK signaling. JNK activation increased BIM phosphorylation at Thr116 that promotes its sequestration by MCL-1 and BCL-2. Notably, a selective and irreversible JNK1-3 inhibitor (JNK-IN-8) reduces BIMThr116 phosphorylation and can release BIM from its sequestration by MCL-1 and BCL-2 to promote apoptosis. Furthermore, use of JNK-IN-8, an MCL-1 antagonist (AZD5991), or their combination in tumor cells lacking PD-L1 is shown to promote apoptosis and reduce long-term clonogenic survival induced by multiple anti-cancer drugs. As confirmation, knockdown of PD-L1 can confer drug resistance in a human colon cancer-derived organoid model that can be reversed by JNK-IN-8. Conclusion Tumor cells with low or absent PD-L1 expression show increased JNK activity that promotes BIM sequestration by MCL-1/BCL-2 to confer multiple drug resistance that can be reversed by a JNK inhibitor. Citation Format: Frank A. Sinicrope, Lei Sun, Arpad Patai, Tara L. Hogenson, Martin E. Fernandez-Zapico, Bo Qin. PD-L1-mediated resistance to chemotherapy is overcome by an irreversible JNK inhibitor in colorectal cancer cells [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 1085.

Abstract Background: PD-L1 is an immune checkpoint protein that may display intrinsic, non-immune functions in human cancer cells. Previously, we discovered that PD-L1 is regulated by BRAFV600E via YAP and c-Jun, and whose deletion can confer chemoresistance due to a decrease in the BH3-only BIM protein in colorectal cancer (CRC) cells. Here, we hypothesize that release of pro-apoptotic BIM from its sequestration by MCL-1 or BCL-2 can overcomes drug resistance mediated by depletion of PD-L1. Materials and Methods: Parental and PD-L1 knockout human (RKO) and murine (MC38) CRC cells with or without reconstituted PD-L1 were utilized. RKO cells with knockdown of MCL-1 or BCL-2 by shRNA were generated. Cells were treated with the MCL-1 selective inhibitor AZD5591 or the BCL-2/BCL-xL inhibitor ABT-263 alone or combined with the MEK inhibitor, cobimetinib. Apoptosis was measured by immunoblotting of cleaved caspase-3 and by Annexin V labeling using FACS. Clonogenic cell survival assay was performed. Protein/protein interaction was analyzed by immunoprecipitation assay. Results: BIM induction by cobimetinib was insufficient to overcome chemoresistance due to loss of PD-L1. Using a potent and selective inhibitor of MCL-1 (AZD-5591), we demonstrate that concurrent antagonism of MCL-1 and MEK can overcome apoptosis resistance in both human and murine PD-L1 knockout CRC cells. Specifically, inhibition of MCL-1 by AZD-5591 or gene knockdown combined with cobimetinib suppresses clonogenic cell survival and enhances apoptosis in cells with PD-L1 knockout, wild type or those with reconstituted PD-L1. Mechanistically, the interaction of BIM with MCL-1 is increased in PD-L1 knockout (versus wild-type cells) in the presence of cobimetinib, and enhanced binding of BIM to MCL-1 is associated with BIM phosphorylation at Thr116. Targeting MCL-1 using AZD-5591 or MCL-1 shRNA releases BIM from its sequestration by MCL-1 and results in enhanced apoptotic signaling induced by cobimetinib to an equivalent extent in wild type and PD-L1 knockout cells with or without reconstitution of PD-L1. Similarly, targeting BCL-2 by ABT-263 or by BCL-2 shRNA releases BIM from its binding to BCL-2, and enhances apoptosis induction by cobimetinib independent of PD-L1 status. Enhanced binding of BIM to BCL-2 was also associated with BIM phosphorylation at Thr116. Conclusion: Chemoresistance mediated by loss of PD-L1 can be reversed by the use of BH3 mimetic drugs combined with MEK inhibition in CRC cells to exploit their apoptotic dependency on BIM expression. Citation Format: Lei Sun, Arpad Patai, Bo Qin, Frank A. Sinicrope. Reversal of PD-L1-mediated chemoresistance by MCL-1/BCL-2 antagonism in colon cancer 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 1821.

STK38, a reader of histone H4 ufmylation, regulates ATM activation.