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Supplementary Table 1

PDF file - 1004K, S1. The Twist box domain is required for full Twist1 transcriptional activity. S2. Quantification of immunofluorescence from isogenic prostate cancer cell lines stably expressing Twist1 and Twist1-F191G. S3. The Twist box domain is required for full Twist1-induced EMT marker phenotypes of prostate cancer cells. S4. Twist1 overexpression induces temporal changes in the material properties of prostate cancer cells during their migration in a wound healing assay. S5. The overexpression of Twist1 or Twist1-F191G does not increase cellular proliferation of prostate cancer cells in vitro. S6. The Twist box domain is required for full Twist1-induced cellular migration in PC3 cells. S7. Twist1 overexpression increases cell traction forces of individual androgenindependent PC3 prostate cancer cells. S8. Twist1 overexpression confers radioresistance to prostate cancer cells which is attenuated by mutation of the Twist box domain. S9. The Twist box domain is required for Twist1-induced soft agar anchorageindependent growth of 22Rv1 prostate cancer cells. S10. Twist1 overexpression does not confer prostate cancer cells increased primary tumorigenicity and slows primary tumor cell growth in vivo. S11. The Twist box domain is required for full Twist1-induced expression of Hoxa9/HOXA9.

Supplementary Figure 1: FGF2 treatment induces branching morphogenesis of primary epithelial cells in 3D culture. Supplementary Figure 2: Basal Expression Levels of TWIST1 in KRAS mutant and MET Amplified/Mutant NSCLC Cell Lines. Supplementary Figure 3: Harmine treatment induces Oncogene-Induced Senescence (OIS) in EGFR and MET-mutant NSCLC cell lines. Supplementary Figure 4: Harmine induces apoptosis in oncogene-driven NSCLC cell lines. Supplementary Figure 5: Harmine treatment promotes TWIST1 degradation and decreases TWIST1 protein stability. Supplementary Figure 6: Silencing of E2A induces apoptosis and phenocopies silencing of TWIST1. Supplementary Figure 7: Overexpression of TWIST1 or its binding partner, E2A, rescues harmine induced growth inhibition. Supplementary Figure 8: Treatment with harmine decreases tumor growth in a KRAS mutant Patient-Derived Xenograph (PDX) model and degrades Twist1 and induces apoptosis in transgenic mouse model of Kras mutant lung cancer. Supplementary Table 1: Rank list of compounds from Connectivity mapping (CMAP) analysis Supplementary Table 2: List of primers used for qRT-PCR Supplementary Table 3: List of primers used for Taqman qRT-PCR Supplementary Table 4: List of antibodies used in current study Supplementary Table 5: Sequences for TWIST1/TCF3 shRNA (5’ â€" 3’) in pKLO.1 Supplementary Table 6: ORFs obtained from Johns Hopkins University HiT Center Supplementary Table 7: Source of Plasmids utilized

PDF file - 116K, Supplemental Table S2 - Myc-CaP + Twist1-F191G versus Myc-CaP + Vector Differentially.

PDF file - 384K, Supplemental Table S1 - Myc-CaP + Twist1 versus Myc-CaP + Vector Differentially.

Supplementary Figure legends and methods

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.

Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) have significantly benefited from the use of EGFR tyrosine kinase inhibitors (TKIs). However, long-term efficacy of these therapies is limited due to de novo resistance (~30%) as well as acquired resistance. Epithelial–mesenchymal transition transcription factors (EMT-TFs), have been identified as drivers of EMT-mediated resistance to EGFR TKIs, however, strategies to target EMT-TFs are lacking. As the third generation EGFR TKI, osimertinib, has now been adopted in the first-line setting, the frequency of T790M mutations will significantly decrease in the acquired resistance setting. Previously less common mechanisms of acquired resistance to first generation EGFR TKIs including EMT are now being observed at an increased frequency after osimertinib. Importantly, there are no other FDA approved targeted therapies after progression on osimertinib. Here, we investigated a novel strategy to overcome EGFR TKI resistance through targeting the EMT-TF, TWIST1, in EGFR-mutant NSCLC. We demonstrated that genetic silencing of TWIST1 or treatment with the TWIST1 inhibitor, harmine, resulted in growth inhibition and apoptosis in EGFR-mutant NSCLC. TWIST1 overexpression resulted in erlotinib and osimertinib resistance in EGFR-mutant NSCLC cells. Conversely, 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 BH3-only gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. As such, pan-BCL2 inhibitor treatment overcame TWIST1-mediated EGFR TKI resistance and were more effective in the setting of TWIST1 overexpression. Finally, in a mouse model of autochthonous EGFR-mutant lung cancer, Twist1 overexpression resulted in erlotinib resistance and suppression of erlotinib-induced apoptosis. These studies establish TWIST1 as a driver of resistance to EGFR TKIs and provide rationale for use of TWIST1 inhibitors or BCL2 inhibitors as means to overcome EMT-mediated resistance to EGFR TKIs.

Therapies for liver cancer particularly those including radiation are still inadequate. Inhibiting the stress response machinery is an appealing anti-cancer and radiosensitizing therapeutic strategy. Heat-shock-protein-90 (HSP90) is a molecular chaperone that is a prominent effector of the stress response machinery and is overexpressed in liver cancer cells. HSP90 client proteins include critical components of pathways implicated in liver cancer cell survival and radioresistance. The effects of a novel non-geldanamycin HSP90 inhibitor, ganetespib, combined with radiation were examined on 3 liver cancer cell lines, Hep3b, HepG2 and HUH7, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γH2AX foci kinetics and client protein expression in pathways important for liver cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined ganetespib-radiation treatment on tumor cell proliferation in a HepG2 hind-flank tumor graft model. Nanomolar levels of ganetespib alone exhibited liver cancer cell anti-cancer activity in vitro as shown by decreased clonogenic survival that was associated with increased apoptotic cell death, prominent G2-M arrest and marked changes in PI3K/AKT/mTOR and RAS/MAPK client protein activity. Ganetespib caused a supra-additive radiosensitization in all liver cancer cell lines at low nanomolar doses with enhancement ratios between 1.33–1.78. These results were confirmed in vivo, where the ganetespib-radiation combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in HepG2 tumor grafts. Our data suggest that combined ganetespib-radiation therapy exhibits promising activity against liver cancer cells, which should be investigated in clinical studies.

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.

Post-translational modifications of histones, such as acetylation and methylation, are differentially positioned in chromatin with respect to gene organization. For example, although histone H3 is often trimethylated on lysine 4 (H3K4me3) and acetylated on lysine 14 (H3K14ac) at active promoter regions, histone H3 lysine 36 trimethylation (H3K36me3) occurs throughout the open reading frames of transcriptionally active genes. The conserved yeast histone acetyltransferase complex, NuA3, specifically binds H3K4me3 through a plant homeodomain (PHD) finger in the Yng1 subunit, and subsequently catalyzes the acetylation of H3K14 through the histone acetyltransferase domain of Sas3, leading to transcription initiation at a subset of genes. We previously found that Ylr455w (Pdp3), an uncharacterized proline-tryptophan-tryptophan-proline (PWWP) domain-containing protein, copurifies with stable members of NuA3. Here, we employ mass-spectrometric analysis of affinity purified Pdp3, biophysical binding assays, and genetic analyses to classify NuA3 into two functionally distinct forms: NuA3a and NuA3b. Although NuA3a uses the PHD finger of Yng1 to interact with H3K4me3 at the 5'-end of open reading frames, NuA3b contains the unique member, Pdp3, which regulates an interaction between NuA3b and H3K36me3 at the transcribed regions of genes through its PWWP domain. We find that deletion of PDP3 decreases NuA3-directed transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Finally, we determine that NuA3a, but not NuA3b, is synthetically lethal in combination with a deletion of the histone acetyltransferase GCN5, indicating NuA3b has a specialized role at coding regions that is independent of Gcn5 activity. Collectively, these studies define a new form of the NuA3 complex that associates with H3K36me3 to effect transcriptional elongation. MS data are available via ProteomeXchange with identifier PXD001156.

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial–mesenchymal transition (EMT) that promotes cancer metastasis. Structure–function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. Implications: Targeting the Twist box domain of Twist1 may effectively limit prostate cancer metastatic potential. Mol Cancer Res; 11(11); 1387–400. ©2013 AACR.

Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the “non-oncogene” addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded “client” proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4–1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies.

Background: Advanced non-small lung cancer (NSCLC) patients with EGFR mutations initially respond to treatment with the EGFR-targeted tyrosine kinase inhibitors (TKIs) such as erlotinib, but will invariably acquire resistance with progression of disease within 10–16 months. Mechanisms of EGFR TKIs resistance include second-site EGFR mutations (>50%), MET amplfication (5–10%), and mutations in PIK3CA ( Methods: To examine the relationship between epithelial plasticity and erlotinib resistance in EGFR mutant lung cancers, we created an inducible CCSP-rtTA/tetO-EgfrL858R/Twist1 (CET) transgenic mice (Twist1 is a key regulator of EMT). We utilized the tetracycline-inducible gene expression system to control EgfrL858R/Twist1 gene expression in the lung by providing or withdrawing doxycycline to the mice. The mice were treated for 3 weeks with erlotinib and scanned by CT each week, and survival of the mice was also recorded. The tumor tissues were collected 1 week and 3 weeks after the start of treatment and used for immunohistochemical staining for H&E, Ki67, and cleaved caspase 3 (CC3). To investigate the mechanisms of treatment resistance, mice lung tumor tissues were collected after 3 weeks' treatment with erlotinib, and tumor lysates were used for RTK signaling antibody array analysis. Results: The lung tumors in the CET mice were more anaplastic than those seen in CE (CCSP-rtTA/tetO-EgfrL858R) mice, with larger, more irregular nuclei. This is consistent with reports of cancers expressing Twist1 being more aggressive. Both genotypes display diffuse hyperplasia instead of discrete tumors, which is similar to the disease seen in EGFR mutant NSCLC in humans. After 3 weeks of erlotinib treatment, a majority of CE mice demonstrated complete and partial responses as well as stable disease. Conversely, over half of the CET mice had tumor progression over the three weeks of treatment. CET mice median overall survival time, from the beginning of treatment, was 6.8 weeks, while CE mice lived a median of 8.7 weeks (p Conclusions: Expression of Twist1 induces resistance to erlotinib in EGFR mutant NSCLC, partially through activation of the Src signaling pathway. Combining erlotinib treatment with the Src inhibitor, dasatinib, in the CET mice leads to tumor regression, prolonged overall survival, as well as decreased proliferation and downstream signaling. Therefore, this combined treatment can potentially overcome Twist1-induced erlotinib resistance in NSCLC. Citation Format: Hailun Wang, Jessica Cades, Zachary Yochum, Katriana Nugent, Charles Rudin, Timothy Burns, Phuoc Tran. Modeling epithelial plasticity-induced erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B47.

Recent advances in the treatment of non-small cell lung cancer (NSCLC) stem from the paradigm shift of classifying patients into subtypes based upon the presence of distinct molecular drivers. Subsets of patients, such as those with EGFR mutations and ALK translocations, have dramatic responses in their tumors to tyrosine kinase inhibitors (TKIs) that specifically inhibit these oncogenic drivers. While many patients initially response to TKIs, therapeutic resistance is inevitable. For EGFR-mutant NSCLC, there are multiple described mechanisms of resistance to EGFR TKIs, including epithelial-mesenchymal transition (EMT). Previous studies have implicated the AXL kinase and ZEB1, an EMT transcription factor (EMT-TF), in EMT-mediated EGFR TKI resistance. We have previously demonstrated that the EMT-TF, TWIST1, is required for oncogene-driven NSCLC tumorigenesis, including those tumors with EGFR mutations. In this study, we investigated the role of TWIST1 in EMT-mediated resistance to EGFR TKIs. We have demonstrated that genetic or pharmacologic inhibition of TWIST1 resulted in growth inhibition in a panel of EGFR-mutant NSCLC cell lines and apoptosis in a subset of these lines. Interestingly, TWIST1 overexpression in EGFR-mutant NSCLC cell lines led to EGFR TKI resistance. Conversely, knockdown of TWIST1 in an erlotinib resistant EGFR-mutant NSCLC cell line restored erlotinib sensitivity. We found that TWIST1 mediates resistance to EGFR TKIs through suppression of apoptosis possibly through decreasing the expression of the pro-apoptotic Bcl-2 member, BCL2L11 (BIM). We observed that TWIST1 knockdown increased BIM levels, while TWIST1 overexpression decreased BIM expression. Furthermore, TWIST1-mediated resistance was overcome by treatment with the BCL-2/BCL-XL inhibitor, ABT-737. Knockdown of BIM recapitulated the resistance seen following TWIST1 overexpression, suggesting that TWIST1 suppression of BIM is a mechanism through which TWIST1 leads to EGFR TKI resistance. To explore the role of TWIST1 in modulating EGFR inhibitor sensitivity in vivo, we used an inducible EGFR-mutant transgenic mouse model, CCSP-rtTA/tetO-EGFRL858R (CE), which expresses EGFRL858R in the lung and a EGFR-mutant/Twist1 transgenic model, CCSP-rtTA/tetO-EGFRL858R/ Twist1- tetO7-luc (CET), which expresses both Twist1 and EGFRL858R in the lung. CET mice had a significantly increased tumor burden, decreased apoptosis and a decreased overall survival compared to CE mice following erlotinib treatment. In summary, we found that TWIST1 overexpression leads to EGFR TKI resistance by suppressing EGFR TKI-induced apoptosis through suppressing BIM expression. Future studies aim to establish the mechanisms of TWIST1 suppression of BIM expression and determine if our TWIST1 inhibitor, harmine, is effective in overcoming EMT-mediated resistance. Citation Format: Zachary A. Yochum, Hailun Wang, Jessica A. Cades, Susheel Khetarpal, Eric H. Huang, Phouc T. Tran, Timothy F. Burns. The EMT transcription factor TWIST1 mediates resistance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4118. doi:10.1158/1538-7445.AM2017-4118

Background: Stabilization of phase II metabolites is an important consideration during bioanalytical method development, method validation and sample analysis. Generic approaches to stabilize these metabolites during storage in liquid-based matrices include pH adjustment of samples prior to storage and/or temperature control; although a variety of other compound-specific stabilization techniques exist. Dried blood spot (DBS) technology is becoming a popular alternative to liquid matrix sampling in many preclinical and clinical applications. However, concerns remain regarding the stability of metabolites stored under ambient conditions using DBS. Results: Experimental data have shown that, under ambient storage conditions, the stability of the glucuronides investigated herein stored as DBS is equivalent to that of liquid samples stored at -80°C. Conclusion: The decision to employ DBS technology for a given study needs to be considered on a case-by-case basis with an understanding of compound-specific metabolism characteristics and clinical study design.

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

Lung cancer is the deadliest cancer worldwide with the majority of cases being non-small cell lung cancer (NSCLC). The most common oncogenic driver in NSCLC patients is mutant KRAS. Mutant KRAS driven proliferation is abated by the failsafe mechanism of oncogene-induced senescence (OIS) in primary cell lines. TWIST1, an inducer of epitheilal-mesenchymal transition (EMT), can overcome OIS, rescuing proliferative capacity of mutant KRAS in primary cells. SNAI1 is another classical EMT master regulator critical in development and is aberrantly expressed in breast, liver, colon, and lung cancers. SNAI1 expression correlates with tumor recurrence and poor patient prognosis. However, there is no evidence for the role of SNAI1 in autochthonous lung tumorigenesis in vivo. To study SNAI1 in lung tumorigenesis, we developed a novel inducible SNAI1 transgenic mouse model. The Club Cell Secretory Protein (CCSP)-rtTA (C) lung tissue specific driver founder line was crossed with a bidirectional transgenic founder line, SNAI1-6SA-tetO7-luc (S), to generate CS mice. CS mice were crossed with inducible oncogenic driver tetO7-KrasG12D (R) mice to produce the triple transgenic, CRS. Inducible SNAI1 expression was confirmed by multiple methods in this novel mouse model. Interestingly, a Kaplan-Meier curve analysis showed that SNAI1 overexpression accelerated KrasG12D-driven lung tumorigenesis compared to KrasG12D alone in vivo. SNAI1 reduced median KrasG12D tumor latency from 31 to 15 weeks of doxycycline (DOX) induction and SNAI1 driven CRS mice developed more aggressive lung adenocarcinomas than the CR model. Upon DOX removal lung tumor moribund mice demonstrated complete radiographic tumor regression, demonstrating tumor maintenance in CRS mice is dependent on KrasG12D and/or SNAI1. To begin to elucidate how SNAI1 may accelerate lung tumorigenesis, inducible SNAI1 mouse embryonic fibroblasts (MEFs) were generated. These MEFs were used to assess if SNAI1 could suppress OIS and promote HRASG12V driven proliferation. While uninduced MEFs senesced within days of HRASG12V infection, DOX induced SNAI1 MEFs had a reduced number of senescence associated Beta-gal positive cells. Furthermore, cellular proliferation showed stagnancy in uninduced HRASG12V infected MEFs, while induced SNAI1 MEFs maintained proliferative capacity. Studies investigating SNAI1-directed epigenetic and transcriptional regulation of metabolic gene targets implicated in circumventing OIS in this model are underway. Overall, these studies demonstrate that SNAI1 may overcome OIS in mutant RAS driven cells to facilitate cellular proliferation and tumorigenesis. Citation Format: Kekoa Taparra, Hailun Wang, Katriana Nugent, Russell Williams, Reem Malek, Jessica Cades, Dean Felsher, Phuoc Tran. Snai1 accelerates Kras driven lung tumorigenesis by overcoming oncogene-induced senescence. [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 2291. doi:10.1158/1538-7445.AM2015-2291

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 frequently observed driver mutation, mutant KRAS. Acquired resistance to therapy targeting other driver mutations (EGFR mutant and ALK translocation positive tumors) is all but inevitable. We recently demonstrated that Twist1, a basic helix-loop-helix transcription factor, 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 in some cases, apoptosis. We have also found that TWIST1 is essential for tumor maintenance in human NSCLCs characterized by oncogenic drivers including KRAS mutation, EGFR mutation and c-MET amplification. As TWIST1 is not typically expressed post-natally, therapies directed against TWIST1 may present a more specific and perhaps less toxic therapy. Targeting the TWIST1 pathway represents an exciting and novel therapeutic strategy which may have a significant clinical impact. We employed a combined bioinformatics-chemical approach to identify pharmacologic inhibitors of TWIST1. We used gene expression profiles from several KRAS mutant human NSCLC cell lines following shRNA-mediated TWIST1 knockdown to perform connectivity map (CMAP) analysis. We found that several of our highly ranked compounds had significant growth inhibitory effects in NSCLC cell lines. Furthermore, several of our candidate agents produced dose-dependent inhibition of TWIST1-induced dissemination in a novel 3D organoid dissemination assay. Interestingly, a family of related harmala alkaloids including harmine ranked highly on the CMAP analysis. We 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. The growth inhibitory effects of the harmala alkaloids correlated with the ability to degrade TWIST1. Additionally, independent of our CMAP analysis, we identified another compound, salinomycin, which appears to modulate cancer cell growth and resulted in a decrease in TWIST1 levels through a post-transcriptional mechanism as well. Interestingly, salinomycin treatment induced a mesenchymal-epithelial transition (MET) in NSCLC which could be partially blocked in TWIST1 overexpressing NSCLC cell lines. We are currently examining the in vivo efficacy of these agents. In conclusion, we have identified several compounds that inhibit TWIST1 dependent dissemination and in some cases can lead to TWIST1 degradation. Since TWIST1 is essential for not only KRAS mutant NSCLC but more broadly for oncogene driven NSCLC, 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 Cades, Neil Neumann, Lucia Mazzacurati, Zachary Yochum, Katriana Nugent, Yoon-Jae Cho, Andrew Ewald, Charles Rudin, Phuoc Tran, Timothy F. Burns. Identification of inhibitors of TWIST1 as a treatment for lung cancer. [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 4771. doi:10.1158/1538-7445.AM2014-4771

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.

The Twist1 gene has diverse roles during development and pathologic states such as cancer. Twist1 is best known for its roles in cancer by inducing an epithelial-mesenchymal transition (EMT) transcriptional program implicated in facilitating tumorigenesis, tumor progression and treatment resistance. Twist1 is a bHLH transcription factor that has both repressor and transactivation functions, but the importance of these different activities for Twist1 cancer phenotypes are unknown. We hypothesized Twist1 may mediate these various functions using distinct structural domains and/or motifs. We disrupted the putative transactivation domain in the Twist box of Twist1 by mutating a critical phenylalanine residue (F191) to glycine. We then created stable isogenic prostate cancer cell lines overexpressing wildtype and F191G versions of Twist1. We assessed the role of the Twist box using in vitro and in vivo assays, which mimic the various stages of cancer progression to metastasis. These include loss of homotypic cell-cell contacts, cell migration and invasion, anoikis resistance and soft agar colony formation. We also observed biophysical cell traction forces on a fabricated substratum and finally performed experimental lung metastasis assays. The overexpression of Twist1 in prostate cancer cells lead to an EMT biomarker phenotype and the F191G mutant lacked expression of some of these markers. The F191G mutant was deficient for transcriptional activity using promoter reporter based assays. Using single cell measurements we found that Twist1 expressing Myc-CaP cells exert more force on the substratum than vector control cells. Additional in vitro assays suggest Twist1 can confer cellular properties associated with increased tumor aggressiveness including increased migration/invasion, cell death/anoikis resistance and in vitro tumorigenic potential by soft agar colony formation. The Twist box mutant, F191G, displayed compromised activity compared to wildtype Twist1 in many of the in vitro assays described above revealing that the Twist box is necessary for many of the pro-metastatic functions of Twist1. We compared the gene expression profile of Twist1 and F191G overexpressing prostate cancer cells by microarray and observed that the F191G mutant had an expression profile that was similar to wildtype Twist1 but attenuated. Lastly, Twist1 overexpression compared to vector control prostate cancer cells showed an increased frequency of metastatic lung tumors using the experimental lung metastasis assay. Interestingly, Twist1 overexpression also resulted in the appearance of extra-thoracic metastases. The F191G mutant was less able to confer prostate cancer cells the ability to colonize metastatic lesions in the lung and resulted in no extra-thoracic metastases. Our results show that F191G mutation behaves as loss of function and is necessary for Twist1-induced metastasis of prostate cancer cells. Citation Format: Rajendra P. Gajula, Sivarajan T. Chettiar, Russell D. Williams, Saravanan Thiyagarajan, Yoshinori Kato, Khaled Aziz, Ruoqi Wang, Nishant Gandhi, Aaron T. Wild, Farhad Vesuna, Jinfang Ma, Tarek Salih, Jessica Cades, Elana Fertig, Shyam Biswal, Timothy F. Burns, Christine Chung, Charles M. Rudin, Venu Raman, Joseph M. Herman, Russell K. Hales, Steven An, Phuoc T. Tran. The Twist box is required for Twist1-induced prostate cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1493. doi:10.1158/1538-7445.AM2013-1493

Purpose: The Twist1 gene has diverse roles during development and pathologic states such as cancer. Twist1 is a bHLH transcription factor that has both repressor and transactivation functions. Twist1 is known to repress transcription by several mechanisms and is therefore considered to mediate its function mainly through transcriptional repression. The Twist1 transactivation domain has been reported but the functional significance of this domain is still unclear. In cancer, Twist1 is best known for its roles in facilitating tumor progression by inducing an epithelial-mesenchymal transition (EMT) transcriptional program implicated in facilitating tumorigenesis, tumor progression and treatment resistance. We hypothesized Twist1 may mediate these various functions using distinct structural domains and/or motifs. Here we have investigated the role of the Twist box domain of Twist1 in prostate cancer. Methods: We disrupted the putative transactivation domain (TD) of Twist1 by mutating a critical phenylalanine residue (F191) to glycine. We then created stable isogenic prostate cancer cell lines overexpressing wildtype and F191G versions of Twist1. We assessed the role of the Twist box using in vitro and in vivo assays, which mimic the various stages of cancer progression to metastasis. These include loss of homotypic cell-cell contacts, cell migration and invasion, anoikis resistance and soft agar colony formation. We also observed single-cell biophysical traction forces on a fabricated substratum and finally performed experimental lung metastasis assays. Results: The overexpression of Twist1 in prostate cancer cells lead to an EMT biomarker phenotype and the F191G mutant lacked expression of some of these markers. The F191G mutant was deficient for transcriptional activity using promoter reporter based assays. Using single cell measurements we found that Twist1 overexpressing prostate cancer cells exert more force on the substratum than vector control cells. Additional in vitro assays suggest Twist1 can confer cellular properties associated with increased tumor cell aggressiveness including increased migration/invasion, cell death/anoikis resistance and in vitro tumorigenic potential by soft agar colony formation. The Twist box mutant, F191G, displayed compromised activity compared to wildtype Twist1 in many of the in vitro assays described above revealing that the Twist box is necessary for many of the pro-metastatic functions of Twist1. We compared the gene expression profile of Twist1 and F191G overexpressing prostate cancer cells by microarray and observed that the F191G mutant had an expression profile that was similar to wildtype Twist1 but attenuated in key gene sets. Lastly, Twist1 overexpression compared to vector control prostate cancer cells showed an increased frequency of metastatic lung tumors using the experimental lung metastasis assay. Interestingly, Twist1 overexpression also resulted in the appearance of extra-thoracic metastases. The F191G mutant was less able to confer prostate cancer cells the ability to colonize metastatic lesions in the lung and resulted in no extra-thoracic metastases. Conclusions: Our results show that the Twist1-F191G mutant behaves as a loss of function and consequently that the Twist1 box is necessary for Twist1-induced metastasis of prostate cancer cells. Citation Format: Rajendra Gajula, Sivarajan Chettiar, Russell Williams, Saravanan Thiyagarajan, Khaled Aziz, Nishant Gandhi, Aaron Wild, Tarek Salih, Yoshinori Kato, Jessica Cades, Elana Fertig, Christine Chung, Joseph Herman, Russell Hales, Charles Rudin, Steven An, Phuoc T. Tran. The Twist box domain is required for Twist1-induced metastasis of prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B45.