Harmala Alkaloid

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Zachary A. Yochum - One of the best experts on this subject based on the ideXlab platform.

  • TWIST1 Suppresses Apoptosis and Mediates Therapeutic Resistance in Non-Small Cell Lung Cancer
    2018
    Co-Authors: Zachary A. Yochum
    Abstract:

    Patients with non-small cell lung cancer (NSCLC) are classified into molecular subgroups based on the presence of oncogenic drivers. Patients with targetable oncogenic drivers, such as mutant EGFR, have benefited from tyrosine kinase inhibitors (TKIs) targeting these oncogenes. However, de-novo and acquired resistance to TKIs limits their efficacy. Studies investigating mechanisms of resistance to TKIs in NSCLC have demonstrated that epithelial-mesenchymal transition (EMT) is associated with resistance. TWIST1 is an EMT-transcription factor that is required for oncogene-driven NSCLC. Utilizing a chemical-bioinformatic screen, we identified the Harmala Alkaloid, harmine, as a first-in-class TWIST1 inhibitor. Harmine inhibited multiple TWIST1 functions, promoted TWIST1 degradation, and had activity in oncogene driver-defined NSCLC cell lines. Additionally, harmine cytotoxicity required degradation of the TWIST1-E2A heterodimer. Harmine also had activity in murine models of KRAS mutant NSCLC. Following identification of this novel TWIST1 inhibitor, we explored TWIST1 as potential target to overcome EGFR TKI resistance in EGFR mutant NSCLC. We demonstrated that TWIST1 expression is sufficient to mediate resistance to EGFR TKIs in vitro and in vivo. Genetic and pharmacological inhibition of TWIST1 in EGFR TKI resistant EGFR mutant cells increased sensitivity to EGFR TKIs. TWIST1-mediated EGFR TKI resistance was due in part to TWIST1 suppression of transcription of the pro-apoptotic gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. In MET-driven NSCLC, TWIST1 overexpression mediated resistance to MET TKIs. Targeting TWIST1 with harmine increased crizotinib sensitivity in MET altered NSCLC cells. We also demonstrated that hepatocyte growth factor (HGF), a known mediator of EGFR and MET TKI resistance, induced TWIST1 expression. Harmine treatment overcame HGF-mediated resistance to MET and EGFR TKIs in MET- and EGFR-driven NSCLC. We also explored the role of TWIST1 in mediating resistance to other targeted agents in NSCLC. We demonstrated that TWIST1 negatively regulates death receptor signaling by directly upregulating transcription of CFLAR (CFLIP), an inhibitor of death receptor 4 and 5. TWIST1 upregulation of cFLIP was associated with resistance to TRAIL-based agents in NSCLC. Overall, these studies demonstrate that targeting TWIST1 is viable therapeutic strategy to overcome resistance to TKIs and TRAIL-based therapies in NSCLC.

  • A First-in-Class TWIST1 Inhibitor with Activity in Oncogene-Driven Lung Cancer
    Molecular cancer research : MCR, 2017
    Co-Authors: Zachary A. Yochum, Jessica Cades, Lucia Mazzacurati, Neil M. Neumann, Susheel K. Khetarpal, Suman Chatterjee, Hailun Wang, Myriam A. Attar, Eric H.-b. Huang, Sarah N. Chatley
    Abstract:

    TWIST1, an epithelial-mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non-small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical-bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a Harmala Alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC.Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764-76. ©2017 AACR.

  • Abstract 3573: TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer
    Molecular and Cellular Biology Genetics, 2016
    Co-Authors: Zachary A. Yochum, Susheel K. Khetarpal, Timothy F. Burns
    Abstract:

    Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. In addition, acquired resistance to currently available targeted therapies is inevitable. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce apoptosis, which suggests that a subset of oncogene dependent NSCLC are potentially “addicted” to TWIST1. Importantly, we have identified the Harmala Alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease levels of TWIST1 and its dimerization partners, the E2A proteins, via degradation. We examined the target genes and pathways required for suppression of apoptosis by TWIST1 and E2A. Genetic or pharmacological (harmine) inhibition of TWIST1 or E2A resulted in apoptosis in several oncogenic driver dependent cell lines. Additionally, treatment with a pan-caspase inhibitor resulted in rescue of growth inhibition following TWIST1 or E2A silencing or harmine treatment. This suggests that apoptosis is the mechanism of growth inhibition following TWIST1 inhibition. TWIST1 or E2A inhibition resulted in increased levels of Bid, Bim, and DR5, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bim and Bid. c-FLIP appears to be a direct transcriptional target of TWIST1 as TWIST1 overexpression leads to transactivation of the c-FLIP promoter and is dependent on the ability of TWIST1 to bind DNA. Interestingly, the TWIST1-E2A heterodimer results in greater promoter transactivation when compared to the TWIST1 homodimer. Furthermore, knockdown of Bim, overexpression of Bcl-2, or overexpression of c-FLIPs resulted in partial rescue of growth inhibition and apoptosis following TWIST1 silencing. However, only knockdown of Bim or Bcl-2 overexpression was able to rescue apoptosis following harmine treatment. This suggests that apoptosis following harmine treatment only requires the intrinsic machinery, while specifically silencing TWIST1 also engages the extrinsic pathway. In summary, we found that the apoptosis observed after TWIST1/E2A inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through its novel target genes, c-FLIP and Bim. Our studies will establish the target genes of TWIST1 that are required for suppression of apoptosis with the ultimate goal of identifying biomarkers of response to TWIST1 inhibitors. We also aim to determine if TWIST1, through its apoptotic target genes, modulates response to targeted therapies or standard chemotherapies. Citation Format: Zachary A. Yochum, Susheel Khetarpal, Timothy F. Burns. TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3573.

  • Abstract 21: TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma
    Molecular and Cellular Biology, 2015
    Co-Authors: Zachary A. Yochum, Phuoc T Tran, Jessica Cades, Sarah N. Chatley, Lucia Mazacurati, Timothy F. Burns
    Abstract:

    A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. The most common oncogene driver mutation is mutant KRAS for which no effective therapies exist. In addition, acquired resistance to currently available targeted therapies for oncogene driver dependent disease is inevitable. Our lab has demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified NSCLC can induce either oncogene induced senescence or apoptosis. The fact that a subset of oncogene dependent NSCLC undergo apoptosis following TWIST1 inhibition suggests that these cells are potentially “addicted” to TWIST1 and might be more vulnerable to TWIST1 inhibitors. Importantly, we have identified the Harmala Alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease TWIST1 levels via degradation. Given that TWIST1 is rarely expressed post-natally, therapies targeting TWIST1 may have minimal toxicities. In the current study, we examine the key TWIST1 functions, target genes and apoptotic pathways that are required for suppression of apoptosis. We found that genetic or pharmacological (harmine) inhibition of TWIST1 resulted in apoptosis in several oncogenic driver dependent cell lines. TWIST1 inhibition resulted in cleavage of caspase 3, 8, 9, and PARP. TWIST1 inhibition resulted in increased levels of Bid, Bim, and TNFRSF10B, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bid. Overexpression of Bcl-2 or c-FLIP resulted in partial abrogation of apoptosis following TWIST1 silencing. These findings suggest that the intrinsic and extrinsic pathways are important for TWIST1 mediated suppression of apoptosis. Preliminary gene expression analysis of NSCLC cells following TWIST1 silencing has identified multiple candidate target genes in these apoptotic pathways. In addition, structure/functional analysis of TWIST1 suggests that nuclear localization, homo- and heterodimerization and proper phosphorylation of TWIST1 are necessary for suppression of apoptosis. Remarkably, TWIST inhibition with harmine treatment decreased tumor growth in our mouse model of KrasG12D/Twist1 NSCLC as well as decreased TWIST1 expression and induced apoptosis. In summary, we found that TWIST1 was required for suppression of apoptosis in several oncogenic driver dependent cell lines. Furthermore, the apoptosis observed after TWIST1 inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through c-FLIP and Bim. Our studies will establish the molecular pathways that are required for suppression of apoptosis with the ultimate goal of identifying predictive biomarkers of response to TWIST1 inhibitors. Citation Format: Zachary A. Yochum, Jessica A. Cades, Lucia Mazacurati, Sarah Chatley, Phuoc T. Tran, Timothy F. Burns. TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 21. doi:10.1158/1538-7445.AM2015-21

  • Abstract 3405: E12 and E47 are essential for TWIST1 dependent suppression of oncogene-induced senescence in NSCLC
    Molecular and Cellular Biology, 2014
    Co-Authors: Lucia Mazzacurati, Phuoc T Tran, Zachary A. Yochum, Sarah N. Chatley, Charles M. Rudin, Timothy F. Burns
    Abstract:

    Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Lung cancer is the leading cause of cancer death in the United States and in the world. Non-small cell lung cancer (NSCLC) is not a single disease entity, but a collection of distinct oncogene driven neoplasms. The most common driver oncogene in NSCLC is mutant-KRAS, which is present in 20-25% of all NSCLC and for which no effective therapies exist. In addition, acquired resistance to the current therapy for the two most frequently observed targetable driver oncogenes in NSCLC, mutant-EGFR and the ALK translocations, is inevitable. Clearly there is a need for novel therapeutic strategies to target these oncogene driven lung cancers. The basic helix-loop-helix (bHLH) transcription factor TWIST1 plays several critical roles in promoting tumorigenesis through inhibition of apoptosis, promotion of metastasis through induction of epithelial-mesenchymal transition (EMT) and inhibition of oncogene-induced senescence (OIS). We recently demonstrated that TWIST1 is essential for tumor maintenance in human NSCLC containing mutant KRAS, mutant EGFR, or amplified cMET. Moreover we have shown that Twist1 cooperates with Kras to induce adenocarcinoma of the lung in mouse models and that inhibition of Twist1 in both a murine model and in human cell lines causes OIS or in some cases apoptosis. We have previously demonstrated that reactivation of OIS after inhibition of TWIST1 occurs independently of the RB/p16, p53/p21 or p27 pathways. In the current study, we examined the role of the TWIST1 binding partners E12 and E47, which are encoded by the E2A locus, in mediating TWIST1 dependent suppression of OIS. E2A encoded proteins have previously been demonstrated to act as tumor suppressors through inhibition of cell proliferation. However, E12 and E47 are overexpressed in several tumor types and this expression can lead to chemoresistance. Furthermore, previous studies have demonstrated that the E12-TWIST1 heterodimerization stabilizes both proteins and in some cases, enhances TWIST1 activity. In our study we have shown that in human KRAS mutant NSCLC cell lines the silencing of the E2A gene products phenocopies the silencing of TWIST1 by inducing either OIS or apoptosis. Furthermore, we have observed significant downregulation of TWIST1 after silencing the E2A gene products. Conversely, overexpression of either E12 or E47 leads to increased TWIST1 protein levels. Interestingly, TWIST1 overexpression leads to E12/E47 stabilization suggesting that heterodimer formation results in a reciprocal stabilization of the binding partner. Finally, we have shown that harmine, a Harmala Alkaloid that leads to degradation of TWIST1, inhibits NSCLC cell line growth and decreases both E12/E47 levels as well. These data suggest that E12/E47 are essential for TWIST mediated suppression of OIS and that targeting of the TWIST1-E12/E47 axis may be an effective therapeutic strategy against oncogene driven NSCLC. Citation Format: Lucia Mazzacurati, Sarah NH Chatley, Zachary Yochum, Charles M. Rudin, Phuoc T. Tran, Timothy F. Burns. E12 and E47 are essential for TWIST1 dependent suppression of oncogene-induced senescence in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2014-3405

Phuoc T Tran - One of the best experts on this subject based on the ideXlab platform.

  • Twist1 is required for the development of UVB-induced squamous cell carcinoma.
    Molecular carcinogenesis, 2021
    Co-Authors: Fernando Eguiarte-solomon, Nicholas Blazanin, Okkyung Rho, Steve Carbajal, Dean W Felsher, Phuoc T Tran, John Digiovanni
    Abstract:

    The transcription factor Twist1 has been reported to be essential for the formation and invasiveness of chemically induced tumors in mouse skin. However, the impact of keratinocyte-specific Twist1 deletion on skin carcinogenesis caused by UVB radiation has not been reported. Deletion of Twist1 in basal keratinocytes of mouse epidermis using K5.Cre × Twist1flox/flox mice led to significantly reduced UVB-induced epidermal hyperproliferation. In addition, keratinocyte-specific deletion of Twist1 significantly suppressed UVB-induced skin carcinogenesis. Further analyses revealed that deletion of Twist1 in cultured keratinocytes or mouse epidermis in vivo led to keratinocyte differentiation. In this regard, deletion of Twist1 in epidermal keratinocytes showed significant induction of early and late differentiation markers, including TG1, K1, OVOL1, loricrin, and filaggrin. Similar results were obtained with topical application of harmine, a Harmala Alkaloid that leads to degradation of Twist1. In contrast, overexpression of Twist1 in cultured keratinocytes suppressed calcium-induced differentiation. Further analyses using both K5.Cre × Twist1flox/flox mice and an inducible system where Twist1 was deleted in bulge region keratinocytes showed loss of expression of hair follicle stem/progenitor markers, including CD34, Lrig1, Lgr5, and Lgr6. These data support the conclusion that Twist1 has a direct role in maintaining the balance between proliferation and differentiation of keratinocytes and keratinocyte stem/progenitor populations. Collectively, these results demonstrate a critical role for Twist1 early in the process of UVB skin carcinogenesis, and that Twist1 may be a novel target for the prevention of cutaneous squamous cell carcinoma.

  • Abstract 21: TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma
    Molecular and Cellular Biology, 2015
    Co-Authors: Zachary A. Yochum, Phuoc T Tran, Jessica Cades, Sarah N. Chatley, Lucia Mazacurati, Timothy F. Burns
    Abstract:

    A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. The most common oncogene driver mutation is mutant KRAS for which no effective therapies exist. In addition, acquired resistance to currently available targeted therapies for oncogene driver dependent disease is inevitable. Our lab has demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified NSCLC can induce either oncogene induced senescence or apoptosis. The fact that a subset of oncogene dependent NSCLC undergo apoptosis following TWIST1 inhibition suggests that these cells are potentially “addicted” to TWIST1 and might be more vulnerable to TWIST1 inhibitors. Importantly, we have identified the Harmala Alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease TWIST1 levels via degradation. Given that TWIST1 is rarely expressed post-natally, therapies targeting TWIST1 may have minimal toxicities. In the current study, we examine the key TWIST1 functions, target genes and apoptotic pathways that are required for suppression of apoptosis. We found that genetic or pharmacological (harmine) inhibition of TWIST1 resulted in apoptosis in several oncogenic driver dependent cell lines. TWIST1 inhibition resulted in cleavage of caspase 3, 8, 9, and PARP. TWIST1 inhibition resulted in increased levels of Bid, Bim, and TNFRSF10B, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bid. Overexpression of Bcl-2 or c-FLIP resulted in partial abrogation of apoptosis following TWIST1 silencing. These findings suggest that the intrinsic and extrinsic pathways are important for TWIST1 mediated suppression of apoptosis. Preliminary gene expression analysis of NSCLC cells following TWIST1 silencing has identified multiple candidate target genes in these apoptotic pathways. In addition, structure/functional analysis of TWIST1 suggests that nuclear localization, homo- and heterodimerization and proper phosphorylation of TWIST1 are necessary for suppression of apoptosis. Remarkably, TWIST inhibition with harmine treatment decreased tumor growth in our mouse model of KrasG12D/Twist1 NSCLC as well as decreased TWIST1 expression and induced apoptosis. In summary, we found that TWIST1 was required for suppression of apoptosis in several oncogenic driver dependent cell lines. Furthermore, the apoptosis observed after TWIST1 inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through c-FLIP and Bim. Our studies will establish the molecular pathways that are required for suppression of apoptosis with the ultimate goal of identifying predictive biomarkers of response to TWIST1 inhibitors. Citation Format: Zachary A. Yochum, Jessica A. Cades, Lucia Mazacurati, Sarah Chatley, Phuoc T. Tran, Timothy F. Burns. TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 21. doi:10.1158/1538-7445.AM2015-21

  • Abstract 3405: E12 and E47 are essential for TWIST1 dependent suppression of oncogene-induced senescence in NSCLC
    Molecular and Cellular Biology, 2014
    Co-Authors: Lucia Mazzacurati, Phuoc T Tran, Zachary A. Yochum, Sarah N. Chatley, Charles M. Rudin, Timothy F. Burns
    Abstract:

    Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Lung cancer is the leading cause of cancer death in the United States and in the world. Non-small cell lung cancer (NSCLC) is not a single disease entity, but a collection of distinct oncogene driven neoplasms. The most common driver oncogene in NSCLC is mutant-KRAS, which is present in 20-25% of all NSCLC and for which no effective therapies exist. In addition, acquired resistance to the current therapy for the two most frequently observed targetable driver oncogenes in NSCLC, mutant-EGFR and the ALK translocations, is inevitable. Clearly there is a need for novel therapeutic strategies to target these oncogene driven lung cancers. The basic helix-loop-helix (bHLH) transcription factor TWIST1 plays several critical roles in promoting tumorigenesis through inhibition of apoptosis, promotion of metastasis through induction of epithelial-mesenchymal transition (EMT) and inhibition of oncogene-induced senescence (OIS). We recently demonstrated that TWIST1 is essential for tumor maintenance in human NSCLC containing mutant KRAS, mutant EGFR, or amplified cMET. Moreover we have shown that Twist1 cooperates with Kras to induce adenocarcinoma of the lung in mouse models and that inhibition of Twist1 in both a murine model and in human cell lines causes OIS or in some cases apoptosis. We have previously demonstrated that reactivation of OIS after inhibition of TWIST1 occurs independently of the RB/p16, p53/p21 or p27 pathways. In the current study, we examined the role of the TWIST1 binding partners E12 and E47, which are encoded by the E2A locus, in mediating TWIST1 dependent suppression of OIS. E2A encoded proteins have previously been demonstrated to act as tumor suppressors through inhibition of cell proliferation. However, E12 and E47 are overexpressed in several tumor types and this expression can lead to chemoresistance. Furthermore, previous studies have demonstrated that the E12-TWIST1 heterodimerization stabilizes both proteins and in some cases, enhances TWIST1 activity. In our study we have shown that in human KRAS mutant NSCLC cell lines the silencing of the E2A gene products phenocopies the silencing of TWIST1 by inducing either OIS or apoptosis. Furthermore, we have observed significant downregulation of TWIST1 after silencing the E2A gene products. Conversely, overexpression of either E12 or E47 leads to increased TWIST1 protein levels. Interestingly, TWIST1 overexpression leads to E12/E47 stabilization suggesting that heterodimer formation results in a reciprocal stabilization of the binding partner. Finally, we have shown that harmine, a Harmala Alkaloid that leads to degradation of TWIST1, inhibits NSCLC cell line growth and decreases both E12/E47 levels as well. These data suggest that E12/E47 are essential for TWIST mediated suppression of OIS and that targeting of the TWIST1-E12/E47 axis may be an effective therapeutic strategy against oncogene driven NSCLC. Citation Format: Lucia Mazzacurati, Sarah NH Chatley, Zachary Yochum, Charles M. Rudin, Phuoc T. Tran, Timothy F. Burns. E12 and E47 are essential for TWIST1 dependent suppression of oncogene-induced senescence in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2014-3405

  • Abstract A12: Screening for TWIST1 inhibitors as a novel therapy for oncogene-driven lung cancer.
    Clinical Cancer Research, 2014
    Co-Authors: Sarah Nh Chatley, Zachary A. Yochum, Lucia Mazzacurati, Neil M. Neumann, Jessica A. Cades, Katriana Nugent, Yoon-jae Cho, Andrew J. Ewald, Charles M. Rudin, Phuoc T Tran
    Abstract:

    A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. Unfortunately, little progress has been made in the treatment of patients with the most frequently observed driver oncogene, mutant KRAS . Furthermore, acquired resistance to the currently targetable driver mutations ( EGFR mutant and ALK translocation positive tumors) is all but inevitable. We recently demonstrated that the basic helix-loop-helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in mouse models and that inhibition of Twist1 in murine models and human lung cancer cell lines led to oncogene induced senescence (OIS) and is some cases, apoptosis. Furthermore, we have found that TWIST1 is essential for tumor maintenance in human NSCLCs characterized by defined oncogenic drivers including KRAS mutation, EGFR mutation and c-MET amplification. As TWIST1 is not typically expressed post-natally, therapies directed against TWIST1 may be a more specific and perhaps less toxic therapy. Therefore, targeting the TWIST1 pathway represents an exciting and novel therapeutic strategy which may have a significant clinical impact. Having identified TWIST1 as a prospective target, we employed a combined bioinformatic-chemical approach with in vitro and in vivo validation to identify pharmacologic inhibitors of TWIST1. We used gene expression profiles from several KRAS mutant human lung cancer cell lines following shRNA-mediated TWIST1 knockdown and from primary KrasG12D/Twist1 mouse tumors to perform connectivity map (CMAP) analysis, in an attempt to identify candidate agents that targeted TWIST1. We have validated the growth inhibitory effects of several of these agents in NSCLC cell lines. Furthermore, using a novel 3D organoid dissemination assay based on primary epithelial tissues from the Twist1 mouse, we demonstrated that several of our candidate agents produced dose-dependent inhibition of TWIST1 induced dissemination. Interestingly, the Harmala Alkaloid, harmine and several other Harmala Alkaloids ranked highly on the CMAP analysis. We have found that harmine could not only inhibit growth in several oncogene driver defined NSCLC cell lines through the induction of apoptosis but could also decrease TWIST1 levels through a post-transcriptional mechanism. Interestingly, the growth inhibitory effects of the Harmala Alkaloids correlated with the ability to degrade TWIST1. We are currently examining the in vivo efficacy of these agents using both xenograft mouse models as well as in our inducible KrasG12D/Twist1 model of lung adenocarcinoma. In conclusion, we have identified several putative inhibitors of TWIST1 through a CMAP analysis and demonstrated that treatment with Harmala Alkaloids leads to induction of apoptosis and degradation of TWIST1 in oncogene driven NSCLC. We have both in vitro and in vivo data suggesting that TWIST1 is not only essential for KRAS mutant NSCLC but more broadly for oncogene driven NSCLC. Therefore, these studies could lead to the development of a novel class of inhibitors which could have a significant clinical impact. Citation Format: Sarah NH Chatley, Jessica A. Cades, Neil M. Neumann, Lucia Mazzacurati, Zachary A. Yochum, Katriana Nugent, Yoon-Jae Cho, Andrew J. Ewald, Charles M. Rudin, Phuoc T. Tran, Timothy F. Burns. Screening for TWIST1 inhibitors as a novel therapy for oncogene-driven lung cancer. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A12.

Timothy F. Burns - One of the best experts on this subject based on the ideXlab platform.

  • Abstract 3573: TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer
    Molecular and Cellular Biology Genetics, 2016
    Co-Authors: Zachary A. Yochum, Susheel K. Khetarpal, Timothy F. Burns
    Abstract:

    Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. In addition, acquired resistance to currently available targeted therapies is inevitable. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce apoptosis, which suggests that a subset of oncogene dependent NSCLC are potentially “addicted” to TWIST1. Importantly, we have identified the Harmala Alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease levels of TWIST1 and its dimerization partners, the E2A proteins, via degradation. We examined the target genes and pathways required for suppression of apoptosis by TWIST1 and E2A. Genetic or pharmacological (harmine) inhibition of TWIST1 or E2A resulted in apoptosis in several oncogenic driver dependent cell lines. Additionally, treatment with a pan-caspase inhibitor resulted in rescue of growth inhibition following TWIST1 or E2A silencing or harmine treatment. This suggests that apoptosis is the mechanism of growth inhibition following TWIST1 inhibition. TWIST1 or E2A inhibition resulted in increased levels of Bid, Bim, and DR5, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bim and Bid. c-FLIP appears to be a direct transcriptional target of TWIST1 as TWIST1 overexpression leads to transactivation of the c-FLIP promoter and is dependent on the ability of TWIST1 to bind DNA. Interestingly, the TWIST1-E2A heterodimer results in greater promoter transactivation when compared to the TWIST1 homodimer. Furthermore, knockdown of Bim, overexpression of Bcl-2, or overexpression of c-FLIPs resulted in partial rescue of growth inhibition and apoptosis following TWIST1 silencing. However, only knockdown of Bim or Bcl-2 overexpression was able to rescue apoptosis following harmine treatment. This suggests that apoptosis following harmine treatment only requires the intrinsic machinery, while specifically silencing TWIST1 also engages the extrinsic pathway. In summary, we found that the apoptosis observed after TWIST1/E2A inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through its novel target genes, c-FLIP and Bim. Our studies will establish the target genes of TWIST1 that are required for suppression of apoptosis with the ultimate goal of identifying biomarkers of response to TWIST1 inhibitors. We also aim to determine if TWIST1, through its apoptotic target genes, modulates response to targeted therapies or standard chemotherapies. Citation Format: Zachary A. Yochum, Susheel Khetarpal, Timothy F. Burns. TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3573.

  • Abstract 21: TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma
    Molecular and Cellular Biology, 2015
    Co-Authors: Zachary A. Yochum, Phuoc T Tran, Jessica Cades, Sarah N. Chatley, Lucia Mazacurati, Timothy F. Burns
    Abstract:

    A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. The most common oncogene driver mutation is mutant KRAS for which no effective therapies exist. In addition, acquired resistance to currently available targeted therapies for oncogene driver dependent disease is inevitable. Our lab has demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified NSCLC can induce either oncogene induced senescence or apoptosis. The fact that a subset of oncogene dependent NSCLC undergo apoptosis following TWIST1 inhibition suggests that these cells are potentially “addicted” to TWIST1 and might be more vulnerable to TWIST1 inhibitors. Importantly, we have identified the Harmala Alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease TWIST1 levels via degradation. Given that TWIST1 is rarely expressed post-natally, therapies targeting TWIST1 may have minimal toxicities. In the current study, we examine the key TWIST1 functions, target genes and apoptotic pathways that are required for suppression of apoptosis. We found that genetic or pharmacological (harmine) inhibition of TWIST1 resulted in apoptosis in several oncogenic driver dependent cell lines. TWIST1 inhibition resulted in cleavage of caspase 3, 8, 9, and PARP. TWIST1 inhibition resulted in increased levels of Bid, Bim, and TNFRSF10B, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bid. Overexpression of Bcl-2 or c-FLIP resulted in partial abrogation of apoptosis following TWIST1 silencing. These findings suggest that the intrinsic and extrinsic pathways are important for TWIST1 mediated suppression of apoptosis. Preliminary gene expression analysis of NSCLC cells following TWIST1 silencing has identified multiple candidate target genes in these apoptotic pathways. In addition, structure/functional analysis of TWIST1 suggests that nuclear localization, homo- and heterodimerization and proper phosphorylation of TWIST1 are necessary for suppression of apoptosis. Remarkably, TWIST inhibition with harmine treatment decreased tumor growth in our mouse model of KrasG12D/Twist1 NSCLC as well as decreased TWIST1 expression and induced apoptosis. In summary, we found that TWIST1 was required for suppression of apoptosis in several oncogenic driver dependent cell lines. Furthermore, the apoptosis observed after TWIST1 inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through c-FLIP and Bim. Our studies will establish the molecular pathways that are required for suppression of apoptosis with the ultimate goal of identifying predictive biomarkers of response to TWIST1 inhibitors. Citation Format: Zachary A. Yochum, Jessica A. Cades, Lucia Mazacurati, Sarah Chatley, Phuoc T. Tran, Timothy F. Burns. TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 21. doi:10.1158/1538-7445.AM2015-21

  • Abstract 3405: E12 and E47 are essential for TWIST1 dependent suppression of oncogene-induced senescence in NSCLC
    Molecular and Cellular Biology, 2014
    Co-Authors: Lucia Mazzacurati, Phuoc T Tran, Zachary A. Yochum, Sarah N. Chatley, Charles M. Rudin, Timothy F. Burns
    Abstract:

    Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Lung cancer is the leading cause of cancer death in the United States and in the world. Non-small cell lung cancer (NSCLC) is not a single disease entity, but a collection of distinct oncogene driven neoplasms. The most common driver oncogene in NSCLC is mutant-KRAS, which is present in 20-25% of all NSCLC and for which no effective therapies exist. In addition, acquired resistance to the current therapy for the two most frequently observed targetable driver oncogenes in NSCLC, mutant-EGFR and the ALK translocations, is inevitable. Clearly there is a need for novel therapeutic strategies to target these oncogene driven lung cancers. The basic helix-loop-helix (bHLH) transcription factor TWIST1 plays several critical roles in promoting tumorigenesis through inhibition of apoptosis, promotion of metastasis through induction of epithelial-mesenchymal transition (EMT) and inhibition of oncogene-induced senescence (OIS). We recently demonstrated that TWIST1 is essential for tumor maintenance in human NSCLC containing mutant KRAS, mutant EGFR, or amplified cMET. Moreover we have shown that Twist1 cooperates with Kras to induce adenocarcinoma of the lung in mouse models and that inhibition of Twist1 in both a murine model and in human cell lines causes OIS or in some cases apoptosis. We have previously demonstrated that reactivation of OIS after inhibition of TWIST1 occurs independently of the RB/p16, p53/p21 or p27 pathways. In the current study, we examined the role of the TWIST1 binding partners E12 and E47, which are encoded by the E2A locus, in mediating TWIST1 dependent suppression of OIS. E2A encoded proteins have previously been demonstrated to act as tumor suppressors through inhibition of cell proliferation. However, E12 and E47 are overexpressed in several tumor types and this expression can lead to chemoresistance. Furthermore, previous studies have demonstrated that the E12-TWIST1 heterodimerization stabilizes both proteins and in some cases, enhances TWIST1 activity. In our study we have shown that in human KRAS mutant NSCLC cell lines the silencing of the E2A gene products phenocopies the silencing of TWIST1 by inducing either OIS or apoptosis. Furthermore, we have observed significant downregulation of TWIST1 after silencing the E2A gene products. Conversely, overexpression of either E12 or E47 leads to increased TWIST1 protein levels. Interestingly, TWIST1 overexpression leads to E12/E47 stabilization suggesting that heterodimer formation results in a reciprocal stabilization of the binding partner. Finally, we have shown that harmine, a Harmala Alkaloid that leads to degradation of TWIST1, inhibits NSCLC cell line growth and decreases both E12/E47 levels as well. These data suggest that E12/E47 are essential for TWIST mediated suppression of OIS and that targeting of the TWIST1-E12/E47 axis may be an effective therapeutic strategy against oncogene driven NSCLC. Citation Format: Lucia Mazzacurati, Sarah NH Chatley, Zachary Yochum, Charles M. Rudin, Phuoc T. Tran, Timothy F. Burns. E12 and E47 are essential for TWIST1 dependent suppression of oncogene-induced senescence in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2014-3405

Susheel K. Khetarpal - One of the best experts on this subject based on the ideXlab platform.

  • A First-in-Class TWIST1 Inhibitor with Activity in Oncogene-Driven Lung Cancer
    Molecular cancer research : MCR, 2017
    Co-Authors: Zachary A. Yochum, Jessica Cades, Lucia Mazzacurati, Neil M. Neumann, Susheel K. Khetarpal, Suman Chatterjee, Hailun Wang, Myriam A. Attar, Eric H.-b. Huang, Sarah N. Chatley
    Abstract:

    TWIST1, an epithelial-mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non-small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical-bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a Harmala Alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC.Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764-76. ©2017 AACR.

  • Abstract 2020: Structure-function analysis of twist1-e2a transcriptional activity in kras-driven non-small cell lung cancer
    Molecular and Cellular Biology Genetics, 2016
    Co-Authors: Susheel K. Khetarpal
    Abstract:

    Lung cancer is the leading cause of cancer death in the United States and in the world. Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1, in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce either oncogene induced senescence or apoptosis. However, the key functions of TWIST1 that are required for its transcriptional activity in cancer are unknown. In the current study, we engineered domain-specific mutations in TWIST1 to determine the impact of altered DNA binding, dimerization and post-translational modifications on its transcriptional activity by luciferase reporter assays. Testing the promoter activity at three TWIST1-regulated loci involved in tumorigenesis, YBX1, SNAI2, and FLIP, we found that DNA binding and nuclear localization were uniformly required for TWIST1 transcriptional function. However, phosphorylation, TWIST1 box function, and ability to form homo- versus hetero- dimers impacted TWIST1 activity in a locus-specific manner. Previous studies have demonstrated that TWIST1 dimerizes with the E2A proteins, E12 and E47. We have shown that silencing of E2A phenocopies loss of TWIST1 in NSCLC and that formation of the TWIST1-E2A heterodimer results in a reciprocal stabilization of the binding partner. Therefore, we sought to determine how differential dimerization by TWIST1 might modulate tumorigenic gene expression. We utilized tethered TWIST1 dimers with either TWIST1 or the E2A proteins to form TWIST1-TWIST1 (homodimer), TWIST1-E12 or -E47 (heterodimers), and tested their transcriptional activities at several TWIST1-regulated promoters. We found that TWIST1-E2A heterodimers could enhance TWIST1 transcriptional activity compared to that of the TWIST-TWIST homodimer. In addition, we determined that the TWIST-E2A heterodimer is degraded by harmine, a Harmala Alkaloid that we have shown degrades TWIST1, whereas the TWIST-TWIST homodimer is resistant to harmine degradation. This suggests that the TWIST1-E2A heterodimer is the key target of harmine. Furthermore, we have found that overexpression of the TWIST1, E2A proteins, or the TWIST1-E2A heterodimer is able to partially rescue harmine induced cytotoxicity in KRAS mutant NSCLC. Taken together, these data suggest that E2A is essential for TWIST1 mediated tumorigenesis and that targeting of the TWIST1-E2A axis may be an effective therapeutic strategy against oncogene-driven NSCLC. Citation Format: Susheel K. Khetarpal. Structure-function analysis of twist1-e2a transcriptional activity in kras-driven non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2020.

  • Abstract 3573: TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer
    Molecular and Cellular Biology Genetics, 2016
    Co-Authors: Zachary A. Yochum, Susheel K. Khetarpal, Timothy F. Burns
    Abstract:

    Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. In addition, acquired resistance to currently available targeted therapies is inevitable. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce apoptosis, which suggests that a subset of oncogene dependent NSCLC are potentially “addicted” to TWIST1. Importantly, we have identified the Harmala Alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease levels of TWIST1 and its dimerization partners, the E2A proteins, via degradation. We examined the target genes and pathways required for suppression of apoptosis by TWIST1 and E2A. Genetic or pharmacological (harmine) inhibition of TWIST1 or E2A resulted in apoptosis in several oncogenic driver dependent cell lines. Additionally, treatment with a pan-caspase inhibitor resulted in rescue of growth inhibition following TWIST1 or E2A silencing or harmine treatment. This suggests that apoptosis is the mechanism of growth inhibition following TWIST1 inhibition. TWIST1 or E2A inhibition resulted in increased levels of Bid, Bim, and DR5, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bim and Bid. c-FLIP appears to be a direct transcriptional target of TWIST1 as TWIST1 overexpression leads to transactivation of the c-FLIP promoter and is dependent on the ability of TWIST1 to bind DNA. Interestingly, the TWIST1-E2A heterodimer results in greater promoter transactivation when compared to the TWIST1 homodimer. Furthermore, knockdown of Bim, overexpression of Bcl-2, or overexpression of c-FLIPs resulted in partial rescue of growth inhibition and apoptosis following TWIST1 silencing. However, only knockdown of Bim or Bcl-2 overexpression was able to rescue apoptosis following harmine treatment. This suggests that apoptosis following harmine treatment only requires the intrinsic machinery, while specifically silencing TWIST1 also engages the extrinsic pathway. In summary, we found that the apoptosis observed after TWIST1/E2A inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through its novel target genes, c-FLIP and Bim. Our studies will establish the target genes of TWIST1 that are required for suppression of apoptosis with the ultimate goal of identifying biomarkers of response to TWIST1 inhibitors. We also aim to determine if TWIST1, through its apoptotic target genes, modulates response to targeted therapies or standard chemotherapies. Citation Format: Zachary A. Yochum, Susheel Khetarpal, Timothy F. Burns. TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3573.

Sarah N. Chatley - One of the best experts on this subject based on the ideXlab platform.

  • A First-in-Class TWIST1 Inhibitor with Activity in Oncogene-Driven Lung Cancer
    Molecular cancer research : MCR, 2017
    Co-Authors: Zachary A. Yochum, Jessica Cades, Lucia Mazzacurati, Neil M. Neumann, Susheel K. Khetarpal, Suman Chatterjee, Hailun Wang, Myriam A. Attar, Eric H.-b. Huang, Sarah N. Chatley
    Abstract:

    TWIST1, an epithelial-mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non-small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical-bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a Harmala Alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC.Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764-76. ©2017 AACR.

  • Abstract 21: TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma
    Molecular and Cellular Biology, 2015
    Co-Authors: Zachary A. Yochum, Phuoc T Tran, Jessica Cades, Sarah N. Chatley, Lucia Mazacurati, Timothy F. Burns
    Abstract:

    A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. The most common oncogene driver mutation is mutant KRAS for which no effective therapies exist. In addition, acquired resistance to currently available targeted therapies for oncogene driver dependent disease is inevitable. Our lab has demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified NSCLC can induce either oncogene induced senescence or apoptosis. The fact that a subset of oncogene dependent NSCLC undergo apoptosis following TWIST1 inhibition suggests that these cells are potentially “addicted” to TWIST1 and might be more vulnerable to TWIST1 inhibitors. Importantly, we have identified the Harmala Alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease TWIST1 levels via degradation. Given that TWIST1 is rarely expressed post-natally, therapies targeting TWIST1 may have minimal toxicities. In the current study, we examine the key TWIST1 functions, target genes and apoptotic pathways that are required for suppression of apoptosis. We found that genetic or pharmacological (harmine) inhibition of TWIST1 resulted in apoptosis in several oncogenic driver dependent cell lines. TWIST1 inhibition resulted in cleavage of caspase 3, 8, 9, and PARP. TWIST1 inhibition resulted in increased levels of Bid, Bim, and TNFRSF10B, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bid. Overexpression of Bcl-2 or c-FLIP resulted in partial abrogation of apoptosis following TWIST1 silencing. These findings suggest that the intrinsic and extrinsic pathways are important for TWIST1 mediated suppression of apoptosis. Preliminary gene expression analysis of NSCLC cells following TWIST1 silencing has identified multiple candidate target genes in these apoptotic pathways. In addition, structure/functional analysis of TWIST1 suggests that nuclear localization, homo- and heterodimerization and proper phosphorylation of TWIST1 are necessary for suppression of apoptosis. Remarkably, TWIST inhibition with harmine treatment decreased tumor growth in our mouse model of KrasG12D/Twist1 NSCLC as well as decreased TWIST1 expression and induced apoptosis. In summary, we found that TWIST1 was required for suppression of apoptosis in several oncogenic driver dependent cell lines. Furthermore, the apoptosis observed after TWIST1 inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through c-FLIP and Bim. Our studies will establish the molecular pathways that are required for suppression of apoptosis with the ultimate goal of identifying predictive biomarkers of response to TWIST1 inhibitors. Citation Format: Zachary A. Yochum, Jessica A. Cades, Lucia Mazacurati, Sarah Chatley, Phuoc T. Tran, Timothy F. Burns. TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 21. doi:10.1158/1538-7445.AM2015-21

  • Abstract 3405: E12 and E47 are essential for TWIST1 dependent suppression of oncogene-induced senescence in NSCLC
    Molecular and Cellular Biology, 2014
    Co-Authors: Lucia Mazzacurati, Phuoc T Tran, Zachary A. Yochum, Sarah N. Chatley, Charles M. Rudin, Timothy F. Burns
    Abstract:

    Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Lung cancer is the leading cause of cancer death in the United States and in the world. Non-small cell lung cancer (NSCLC) is not a single disease entity, but a collection of distinct oncogene driven neoplasms. The most common driver oncogene in NSCLC is mutant-KRAS, which is present in 20-25% of all NSCLC and for which no effective therapies exist. In addition, acquired resistance to the current therapy for the two most frequently observed targetable driver oncogenes in NSCLC, mutant-EGFR and the ALK translocations, is inevitable. Clearly there is a need for novel therapeutic strategies to target these oncogene driven lung cancers. The basic helix-loop-helix (bHLH) transcription factor TWIST1 plays several critical roles in promoting tumorigenesis through inhibition of apoptosis, promotion of metastasis through induction of epithelial-mesenchymal transition (EMT) and inhibition of oncogene-induced senescence (OIS). We recently demonstrated that TWIST1 is essential for tumor maintenance in human NSCLC containing mutant KRAS, mutant EGFR, or amplified cMET. Moreover we have shown that Twist1 cooperates with Kras to induce adenocarcinoma of the lung in mouse models and that inhibition of Twist1 in both a murine model and in human cell lines causes OIS or in some cases apoptosis. We have previously demonstrated that reactivation of OIS after inhibition of TWIST1 occurs independently of the RB/p16, p53/p21 or p27 pathways. In the current study, we examined the role of the TWIST1 binding partners E12 and E47, which are encoded by the E2A locus, in mediating TWIST1 dependent suppression of OIS. E2A encoded proteins have previously been demonstrated to act as tumor suppressors through inhibition of cell proliferation. However, E12 and E47 are overexpressed in several tumor types and this expression can lead to chemoresistance. Furthermore, previous studies have demonstrated that the E12-TWIST1 heterodimerization stabilizes both proteins and in some cases, enhances TWIST1 activity. In our study we have shown that in human KRAS mutant NSCLC cell lines the silencing of the E2A gene products phenocopies the silencing of TWIST1 by inducing either OIS or apoptosis. Furthermore, we have observed significant downregulation of TWIST1 after silencing the E2A gene products. Conversely, overexpression of either E12 or E47 leads to increased TWIST1 protein levels. Interestingly, TWIST1 overexpression leads to E12/E47 stabilization suggesting that heterodimer formation results in a reciprocal stabilization of the binding partner. Finally, we have shown that harmine, a Harmala Alkaloid that leads to degradation of TWIST1, inhibits NSCLC cell line growth and decreases both E12/E47 levels as well. These data suggest that E12/E47 are essential for TWIST mediated suppression of OIS and that targeting of the TWIST1-E12/E47 axis may be an effective therapeutic strategy against oncogene driven NSCLC. Citation Format: Lucia Mazzacurati, Sarah NH Chatley, Zachary Yochum, Charles M. Rudin, Phuoc T. Tran, Timothy F. Burns. E12 and E47 are essential for TWIST1 dependent suppression of oncogene-induced senescence in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2014-3405

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