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Advanced lung cancer patients that present with activating mutations in the epidermal growth factor receptor (EGFR) are treated with EGFR tyrosine kinase inhibitors (EGFR TKIs). While initially effective, all patients eventually develop therapeutic resistance and experience disease progression. Strategies to prevent EGFR TKI resistance are needed to improve patient outcomes. We chronically exposed H1975 cells (EGFR-L858R/T790M) to EGFR TKI osimertinib to study emergence of resistance. H1975 cells that were expanded under drug treatment (designated H1975OR) acquired an EMT phenotype and were re-sensitized to EGFR TKI upon prolonged drug withdrawal. These features led us to classify H1975OR as a model of drug tolerance rather than a model of stable drug resistance. Bulk RNA-sequencing revealed significant dysregulation of chromatin modifying genes in H1975OR. Bromodomain containing protein 9 (BRD9) was among the set of significantly upregulated chromatin regulators and was selected for further investigation as a mediator of drug tolerance. Although BRD9 is known to control stemness and EMT, its role in promoting EMT-associated TKI resistance is unknown. To test the contribution of BRD9 to EGFR TKI tolerance, pharmacological inhibition of BRD9 by the selective inhibitor, I-BRD9 significantly increased sensitivity to TKI in models with EMT phenotypes (IC50 reduced 3-4 fold). In contrast, I-BRD9 did not affect TKI sensitivity in two models where EGFR TKI resistance was genetically fixed. To gain mechanistic insights, H1975OR cells were treated with osimertinib +/-I-BRD9 and subjected to RNA-sequencing. Combination of osimertinib with I-BRD9 resulted in a significant decrease in EMT-related genes, including MMP9, Zeb2, PDGFRb and IL6. Further, use of I-BRD9 in these models diminished the mesenchymal phenotype, as measured in cell invasion assays. Genetic knockdown of BRD9 via shRNA phenocopied effects of I-BRD9 treatment, supporting BRD9 as the therapeutic target of I-BRD9 in our cell line models. To further establish a role for BRD9 in the emergence of drug tolerant cells, we exposed treatment naïve H1975 cells to EGFR TKI ± I-BRD9. I-BRD9 significantly decreased the size of the EGFR TKI tolerant population. In time course studies, I-BRD9 also delayed onset of TKI resistance. In conclusion, our data identifies BRD9 as a novel mediator of EMT-associated EGFR-TKI tolerance in EGFR-mutant lung cancer. Our data further implicates BRD9 inhibition as a novel strategy to delay the emergence of drug tolerant cells that eventually give rise to stable drug resistance. This work was supported by the Roswell Park Alliance Foundation and National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Comprehensive Cancer Center’s Genomics and Bioinformatics Shared Resources. Citation Format: Hannah Calkins, Tatiana Shaurova, David W. Goodrich, Mukund Seshadri, Candace S. Johnson, Pamela A. Hershberger. BRD9 inhibition overcomes epithelial to mesenchymal transition (EMT)-associated tyrosine kinase inhibitor (TKI) tolerance in epidermal growth factor receptor (EGFR) mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1093.

Estrogen receptor (ER) agonists and antagonists elicit distinct responses in non–small cell lung cancer (NSCLC) cells. To determine how such responses are generated, the expression of ERα, ERβ, and ER coregulators in human lung fibroblasts and human NSCLC cell lines was evaluated by immunoblot. Ligand-dependent estrogenic responses in NSCLC cells are probably generated via ERβ and the p160 coactivator GRIP1/TIF2, because expression of these proteins was detected, but not full-length ERα or the p160 coactivator SRC-1. ERβ and GRIP1/TIF2 are shown to interact in vitro in a ligand-dependent manner and thus may form functional transcription complexes in NSCLC cells. Furthermore, the capacity of ER ligands to regulate gene expression in NSCLC cells was explored using gene miniarrays. Expression profiles were examined after treatment with ER agonist 17-β-estradiol (E2), the pure ER antagonist ICI 182,780 (fulvestrant, Faslodex), or epidermal growth factor, which served as a positive control for an alternative growth stimulus. E-cadherin and inhibitor of differentiation 2 were differentially regulated by E2 versus ICI 182,780 in 201T and 273T NSCLC cell lines. Epidermal growth factor also stimulated proliferation of these cells but had no effect on expression of E-cadherin and inhibitor of differentiation 2, suggesting they are specific targets of ER signaling. These data show that NSCLC cells respond to estrogens/antiestrogens by altering endogenous gene expression and support a model in which ICI 182,780 reduces proliferation of NSCLC cells via its ability to disrupt ER signaling. ICI 182,780 may therefore have therapeutic benefit in NSCLC.

Background. Despite remarkable initial therapeutic responses, most patients develop resistance to the first generation of EGFR tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, within one year of treatment initiation. Epithelial-mesenchymal transition (EMT) is one of the key mechanisms of resistance to EGFR TKIs. We recently demonstrated that vitamin D promotes epithelial phenotype in NSCLC cells. We hypothesized that we could prevent/overcome EMT-driven resistance by combining vitamin D with EGFR TKIs. Methods. EGFR-mutant, erlotinib sensitive HCC827 cells were treated with: vehicle control, 100nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 1ng/ml TGF-β, or 1,25(OH)2D3+TGF-β for 14 days. Expression of EMT markers was determined by q-RT PCR and immunoblot. Sensitivity to erlotinib was analyzed by MTT assay. For the in-vivo study, HCC 827 tumor xenografts were established in vitamin-D deficient mice. Mice were stratified based on tumor volume to receive diets containing 25 IU vitamin D3/kg (deficient) or 10,000 IU vitamin D3/kg (supplemented). Erlotinib treatment (12.5 mg/kg) was initiated two weeks after the diet switch and administered 5 days per week for the duration of the study. Tumors were collected two weeks after the diet switch (baseline), 48 hours after the erlotinib initiation (acute response) and upon treatment failure (outgrowth). Results. Vitamin D attenuated expression of mesenchymal markers in the presence of TGF-β in-vitro and opposed TGF-β driven resistance to erlotinib. Erlotinib EC50 values were 9.85 nM, 122 nM and 3.83 nM for vehicle, TGF-β, and 1,25(OH)2D3+TGF-β treated cells, respectively. In-vivo, vitamin D supplementation enhanced acute response to erlotinib. We documented a greater decrease in tumor volume after 48h of erlotinib initiation (1.1% of baseline ±19.3% in vitamin D deficient diet and 25.9% ±29.5% in supplemented diet). Greater efficacy was accompanied by increased suppression of STAT3 phosphorylation and decreased vimentin expression in the vitamin D supplemented group. Although we observed a trend towards a longer progression-free survival in animals maintained on vitamin D supplemented diet (8 vs 5.5 weeks), statistical significance was not achieved. The outgrowth tumors in both groups maintained their epithelial phenotype but exhibited a significant increase in the expression of AXL tyrosine kinase. However, outgrowth tumors from the vitamin D supplemented group had lower AXL expression than those from the vitamin D deficient group. Conclusion. Vitamin D supports epithelial phenotype in-vitro and enhances acute response to erlotinib in-vivo, possibly, by suppressing STAT3 signaling. Vitamin D supplementation also attenuates overexpression of AXL tyrosine kinase in erlotinib resistant tumors. The clinical implication of our work is that patients diagnosed with EGFR mutant NSCLC who receive erlotinib may benefit from vitamin D supplementation. Citation Format: Tatiana Shaurova, Suzanne F. Shoemaker, Pamela A. Hershberger. Vitamin D enhances erlotinib response in EGFR-mutant 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 4674.

Introduction 25-hydroxyvitamin D3 (25(OH)D3) levels are associated with increased overall survival in early stage non-small cell lung cancer (NSCLC). Studies suggest that 25(OH)D3 is converted locally in tissues by CYP27B1 into the active metabolite, 1,25-dihydroxyvitamin D (1α,25(OH)2D3). Once produced, 1α,25(OH)2D3 exerts anti-tumor effects by binding to the vitamin D receptor (VDR) and regulating target gene expression. It is unknown whether CYP27B1 is necessary for NSCLC cells to respond to 25(OH)D3. We therefore tested the requirement for VDR and CYP27B1 in mediating the anti-tumor effects of 25(OH)D3 in NSCLC. Methods Immunoblot and qRT-PCR were used to evaluate VDR expression. Small interfering RNA was used to knock down the VDR. Colony formation assays and qRT-PCR were used to assess effects of 25(OH)D3 and 1α,25(OH)2D on growth and transcriptional responses. To study the requirement for CYP27B1, pharmacological inhibition was achieved using ketoconazole, and 1α,25(OH)2D3 production was measured by LC/MS-MS. A xenograft study was performed to determine the in vivo effect of modulating 25(OH)D3 levels. Results NSCLC cells expressing high levels of VDR (VDRhigh) are more responsive to vitamin D metabolites. In these cells, 25(OH)D3 and 1α,25(OH)2D3 induce target gene transcription and inhibit colony formation. These effects are not observed in VDRlow cells. When VDRhigh cells were transfected with VDR siRNA, the ability of 25(OH)D3 to induce target gene transcription was diminished, and no VDR protein was observed. VDRhigh cells were exposed to ketoconazole to determine if effects of 25(OH)D3 required conversion to 1α,25(OH)2D3. Ketoconazole effectively abrogated production of 1α,25(OH)2D3 from 25(OH)D3. However, VDR target gene expression remained induced by 25(OH)D3. Thus, NSCLC cells may not require CYP27B1 to promote vitamin D signaling. To more specifically inhibit CYP27B1, zinc finger nucleases (ZFNs) were used to create CYP27B1 knockout cells. The ZFNs have been transfected into cells, assays optimized, and positive clones are being expanded. Lastly, a mouse xenograft experiment was performed to determine if 25(OH)D3 promotes a decrease in tumor volume. Mice were fed diets containing 100, 1,000 or 10,000 IU/kg vitamin D3 prior to implantation of VDRhigh NSCLC cells. Mice fed the 10,000 IU/kg diet had a statistically significant decrease in tumor volume. Conclusions NSCLC cells expressing high levels of VDR display increased responsiveness to vitamin D metabolites. Although NSCLC cells express CYP27B1, our results demonstrate that the enzyme may not be required to achieve vitamin D signaling. Rather, 25(OH)D3 may act via the VDR to elicit an anti-tumor responses. The implication of these findings is that dietary supplementation to increase circulating 25(OH)D3 may be beneficial in a subset of NSCLC patients. Funding provided by NIH RO1 CA132844, training grant 5T32CA009072-37 and P30CA47904 Citation Format: Alissa R. Verone, Suzanne Shoemaker, Robert Parise, Jan H. Beumer, Pamela A. Hershberger. 25-hydroxyvitamin D3 induces vitamin D signaling independent of CYP27B1 in non-small cell lung cancer cells. [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 4751. doi:10.1158/1538-7445.AM2014-4751

Progress has recently been made in identifying populations at risk for lung cancer using genetic, clinical and demographic information. The N-nitroso-tris-chloroethylurea (NTCU) mouse model, in which animals develop premalignant histopathology similar to that seen in humans, is used to examine the potential efficacy of chemoprevention agents to be utilized in at risk populations. We identified 25 µl of 40 mM NTCU /week as the optimal dosing regimen in SWR/J mice for chemoprevention studies. At this dose, topical treatments of NTCU induce predominantly low-grade dysplastic lesions by 15 weeks (w) and high-grade dysplastic (HGD) lesions by 25 w in the large airways. Additionally we found that NTCU stimulates a state of chronic inflammation associated with the development of dysplasias. Epidemiologic studies indicate that there is an inverse relationship between vitamin D and the risk and prognosis of lung cancer. Vitamin D acts through the vitamin D receptor to promote cellular differentiation and inhibit proliferation and inflammation. The effect of vitamin D on cancer progression was tested in the NTCU model, using dietary vitamin D3 (0 or 2000 IU/kg) alone and in combination with intraperitoneal injections of the active metabolite of vitamin D, calcitriol (80 ug/kg). Female mice were randomized to 6 treatment groups (n=15 mice/group/time point (15 and 25 w)). Disease was evaluated by enumerating the percentage of HGD lesions, per the total area in serial H&E sections of the lung. The percentage of HGD lesions in the large airways was reduced in the vitamin D sufficient mice (SN) (8.72%, P Furthermore, there was a significant increase in proliferation associated with increased HGD. Following 25 w of NTCU treatment there was a 2-fold increase in Ki-67 staining all groups compared all groups after 15 w of NTCU. However, the was 30% more Ki-67staining in the DN (P Citation Format: Sarah A. Mazzilli, Mary E. Reid, Paul N. Bogner, Kristopher Attwood, Pamela A. Hershberger, Donald L. Trump, Candace S. Johnson. Vitamin D sufficiency slows the progression of dysplasic lesions in the NTCU mouse model of lung squamous cell carcinoma. [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 238. doi:10.1158/1538-7445.AM2014-238

Introduction: TMZ is a non-classical alkylating agent that induces DNA damage leading to cytotoxicity, with modest clinical activity in metastatic melanoma. DNA repair modulation with PARP inhibitors enhances TMZ cytotoxicity in vitro and in vivo, and this strategy is being investigated in clinical trials. We hypothesized that HDAC inhibition with SAHA would lead to increased DNA damage and potentiate the effect of the PARP inhibitor MK-4827 on TMZ cytotoxicity. Methods: Human melanoma cell lines A375 and FEMX-1 were used. Cells were treated in the absence or presence of TMZ, SAHA, and MK-4827 at the indicated concentrations for 72 hr in 96-well plates. Cell viability was determined using MTS assays and normalized to vehicle-treated control cells. Drugs were used at the final concentrations indicated in Table 1. Results: MK-4827 at 10 nM increased the cytotoxicity of TMZ in FEMX-1 but not in A375 cells, however 100 and 200 nM MK-4827 moderately potentiated TMZ cytotoxicity in both cell lines. The combination of TMZ with 1 µM SAHA and 200 nM MK-4827 resulted in profound cytotoxicity at reduced doses of TMZ (0.2 mM in FEMX-1 and 1.0 mM in A375), much lower than TMZ alone or in combination with either SAHA or MK-4827. >80% cell kill was achieved in A375 and >95% in FEMX-1 near the IC50 concentration of TMZ and at clinically achievable concentrations. Conclusion: PARP inhibition offers a strategy to reverse melanoma TMZ resistance. The concomitant inhibition of HDAC with SAHA and of PARP with MK-4827 profoundly enhanced TMZ cytotoxicity at clinically achievable doses of all 3 agents. We are presently optimizing the dosing schedules of the 3 drugs in preclinical models and investigating the mechanism(s) underlying this potentiation. These findings support the investigation of the triple drug combination (TMZ+SAHA+MK-4827) for metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5499. doi:10.1158/1538-7445.AM2011-5499

Introduction Cytidine drugs, such as gemcitabine, undergo rapid, inactivating catabolism by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine drug metabolism. However, p.o. THU is at most 20% bioavailable (Beumer et al.), which limits its preclinical evaluation and clinical use. Therefore, the more lipophilic prodrug triacetyl THU (taTHU) was developed. We characterized THU pharmacokinetics after administration of taTHU to mice. Methods Mice were dosed 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters and p.o. bioavailability were calculated non-compartmentally and compartmentally. We assessed taTHU inhibition of metabolism of gemcitabine by recombinant CD. ResultsTHU parameterstaTHU i.v. 150 mg/kgtaTHU p.o. 150 mg/kgCmax (µg/mL)10611.8Tmax (min)560Half-life (min)235122AUC0-inf (mg/mL*min)7.553.36Vd/F (L/kg)4.495.26Cl/F (mL/min/kg)13.229.8F-THUi.v. (%)68.030.3% dose in 0-16 h urine405.6% dose in 0-24 h urine5512 THU, after 150 mg/kg taTHU i.v., had a 235 min terminal half-life and produced plasma THU concentrations >1 µg/mL, the concentration shown to inhibit CD, for 10 h. A multi-compartment model fit the data best. 150 mg/kg p.o. taTHU produced a concentration versus time profile with a plateau of approximately 10 µg/mL from 0.5-2 h, followed by a decline with a 122 min half-life. Approximately 68% of i.v. taTHU was converted to THU. Approximately 30% of p.o. taTHU reached the systemic circulation as THU. After i.v dosing, renal excretion accounted for 40-55% of the taTHU dose, 26-35% as THU and an additional 14-19% as acetylated forms of THU. After p.o. dosing, the renal excretion percentages were 6-12%, 5.2-11%, and 0.42-0.71%, respectively. taTHU did not inhibit recombinant CD. Conclusion The availability of THU after p.o. taTHU in mice is 30%, as compared to the 20% achieved after p.o. THU. Clinical studies are warranted to evaluate availability of THU from taTHU in humans. Support: N01-CM07106, N01-CM52202, P30-CA47904 from the NCI and P41-EB001978. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4549.

Introduction: We previously reported that the vitamin D3 catabolizing enzyme CYP24 is over-expressed in non-small cell lung cancer (NSCLC) cells, where it decreases the growth inhibitory effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Studies were conducted to establish the clinical relevance of this finding and the mechanism for CYP24 over-expression in NSCLC cells. Methods: CYP24 mRNA expression was quantified by real-time PCR in patient-matched histologically normal lung tissue and primary lung tumors. To elucidate factors that control CYP24 expression, a panel of NSCLC cell lines that exhibit a 70-fold variation in CYP24 mRNA levels under basal growth conditions was used (relative mRNA expression H292 =1.0; 201T = 7.0; 128.88T = 68; A549 =69). CYP24 gene copy number was assessed by fluorescence in situ hybridization. CYP24 promoter activity was measured following transfection of NSCLC cells with either a wildtype promoter-luciferase reporter plasmid or plasmids harboring CYP24 promoter mutations. Transcription factor binding to CYP24 promoter elements was evaluated by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assays. Results: CYP24 was significantly over-expressed in tumors (p = 0.0017), with 53% of NSCLC cases showing a minimum of 5-fold over-expression. CYP24 gene copy number was comparable among the NSCLC cell lines used. When NSCLC cells were transfected with a wildtype CYP24 promoter-luciferase reporter plasmid, significant differences in promoter activity were observed that paralleled endogenous CYP24 transcript levels. ChIP assays revealed that the CCAAT-enhancer binding protein C/EBPβ binds to the endogenous CYP24 promoter under basal growth conditions in 128.88T cells. To establish a role for C/EBP proteins in regulating CYP24 transcription, we introduced mutations into the C/EBP binding site within the CYP24 promoter. The mutations disrupted C/EBP binding and significantly reduced basal promoter activity in 128.88T and A549 cells. EMSAs that employed a consensus C/EBP site as probe demonstrated that C/EBP binding increases with CYP24 promoter activity: nuclear extracts from untreated H292 and 201T cells contained low levels of C/EBP binding activity, whereas 128.88T and A549 extracts contained high levels. Conclusions: CYP24 mRNA is frequently over-expressed in primary lung tumors. A C/EBPβ-mediated transcriptional control mechanism contributes to the increased expression of CYP24 in NSCLC cells. Signaling pathways that increase C/EBPβ expression/activation may contribute to lung cancer by upregulating CYP24 and allowing neoplastic cells to bypass the antiproliferative effects of 1,25(OH)2D3. This work was supported by the University of Pittsburgh Center for Environmental Oncology, the Hillman Foundation, the Pittsburgh Foundation and R01 CA132844. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4973.