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Supplementary Figure 3

Supplementary Table 2

Supplementary figure 3 shows combining trametinib with different classes of HDAC inhibitors in COLO 201 cells

The EGFR/RAS/MEK/ERK signaling pathway (ERK/MAPK) is hyperactivated in most colorectal cancers. A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in colorectal cancer cells. Nevertheless, these drugs do induce expression of proapoptotic factors, suggesting they may prime colorectal cancer cells to undergo apoptosis. As histone deacetylase inhibitors (HDACis) induce expression of multiple proapoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger colorectal cancer cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in colorectal cancer cell lines and patient-derived tumor organoids in vitro, and attenuated Apc-initiated adenoma formation in vivo. Mechanistically, combined MAPK/HDAC inhibition enhanced expression of the BH3-only proapoptotic proteins BIM and BMF, and their knockdown significantly attenuated MAPK/HDAC inhibitor–induced apoptosis. Importantly, we demonstrate that the paradigm of combined MAPK/HDAC inhibitor treatment to induce apoptosis can be tailored to specific MAPK genotypes in colorectal cancers, by combining an HDAC inhibitor with either an EGFR, KRASG12C or BRAFV600 inhibitor in KRAS/BRAFWT; KRASG12C, BRAFV600E colorectal cancer cell lines, respectively. These findings identify a series of ERK/MAPK genotype-tailored treatment strategies that can readily undergo clinical testing for the treatment of colorectal cancer.

Supplementary table 1

The EGFR/RAS/MEK/ERK signaling pathway (ERK/MAPK) is hyperactivated in most colorectal cancers. A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in colorectal cancer cells. Nevertheless, these drugs do induce expression of proapoptotic factors, suggesting they may prime colorectal cancer cells to undergo apoptosis. As histone deacetylase inhibitors (HDACis) induce expression of multiple proapoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger colorectal cancer cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in colorectal cancer cell lines and patient-derived tumor organoids in vitro, and attenuated Apc-initiated adenoma formation in vivo. Mechanistically, combined MAPK/HDAC inhibition enhanced expression of the BH3-only proapoptotic proteins BIM and BMF, and their knockdown significantly attenuated MAPK/HDAC inhibitor–induced apoptosis. Importantly, we demonstrate that the paradigm of combined MAPK/HDAC inhibitor treatment to induce apoptosis can be tailored to specific MAPK genotypes in colorectal cancers, by combining an HDAC inhibitor with either an EGFR, KRASG12C or BRAFV600 inhibitor in KRAS/BRAFWT; KRASG12C, BRAFV600E colorectal cancer cell lines, respectively. These findings identify a series of ERK/MAPK genotype-tailored treatment strategies that can readily undergo clinical testing for the treatment of colorectal cancer.

Epigenetics; Tumour-suppressor proteins Epigenética; Proteínas supresoras de tumores Epigenètica; Proteïnes supresores de tumors Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC. This project was supported by NHMRC project grant (1107831), a Cancer Council Victoria Grant (1164674) and the Operational Infrastructure Support Programme, Victorian Government, Australia. JMM was supported by a NHMRC Senior Research Fellowship (1046092). IYL was supported by F J Fletcher Research Scholarship and Randal and Louisa Alcock Scholarship from the University of Melbourne. LJJ was supported by La Trobe University Australian Postgraduate Awards. IN was supported by La Trobe University Postgraduate Research Scholarship. JWTT was supported by the University of Melbourne Australian Postgraduate Awards. OMS is a National Health and Medical Research Council (NHMRC) Senior Research Fellow (APP1136119). Open Access funding enabled and organized by CAUL and its Member Institutions.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with treatment limited to Cisplatin and Pemetrexed chemotherapy. Recently, we showed that drugs targeting the BCL-2-regulated apoptosis pathway could kill MPM cell lines in vitro, and control tumor growth in vivo. These studies showed BCL-XL was the dominant pro-survival BCL-2 family member correlating with its high-level expression in cells and patient tumor samples. In this study we show another inhibitor, AZD4320 that targets BCL-XL (and BCL-2), can also potently kill MPM tumor cells in vitro (EC50 values in the 200 nM range) and this effect is enhanced by co-inhibition of MCL-1 using AZD5991. Moreover, we show that a novel nanoparticle, AZD0466, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer, was as effective as standard-of-care chemotherapy, Cisplatin, at inhibiting tumor growth in mouse xenograft studies, and this effect was enhanced when both drugs were combined. Critically, the degree of thrombocytopenia, an on-target toxicity associated with BCL-XL inhibition, was significantly reduced throughout the treatment period compared to other BCL-XL-targeting BH3-mimetics. These pre-clinical findings provide a rationale for the future clinical evaluation for novel BH3-mimetic formulations in MPM, and indeed, other solid tumor types dependent on BCL-XL.

Amplification or overexpression of the FGFR family of receptor tyrosine kinases occurs in a significant proportion of gastric cancers. Regorafenib is a multikinase inhibitor of angiogenic and oncogenic kinases, including FGFR, which showed activity in the randomized phase II INTEGRATE clinical trial in advanced gastric cancer. There are currently no biomarkers that predict response to this agent, and whether regorafenib is preferentially active in FGFR-driven cancers is unknown. Through screening 25 gastric cancer cell lines, we identified five cell lines that were exquisitely sensitive to regorafenib, four of which harbored amplification or overexpression of FGFR family members. These four cell lines were also sensitive to the FGFR-specific inhibitors, BGJ398, erdafitinib, and TAS-120. Regorafenib inhibited FGFR-driven MAPK signaling in these cell lines, and knockdown studies confirmed their dependence on specific FGFRs for proliferation. In the INTEGRATE trial cohort, amplification or overexpression of FGFRs 1–4 was detected in 8%–19% of cases, however, this was not associated with improved progression-free survival and no objective responses were observed in these cases. Further preclinical analyses revealed FGFR-driven gastric cancer cell lines rapidly reactivate MAPK/ERK signaling in response to FGFR inhibition, which may underlie the limited clinical response to regorafenib. Importantly, combination treatment with an FGFR and MEK inhibitor delayed MAPK/ERK reactivation and synergistically inhibited proliferation of FGFR-driven gastric cancer cell lines. These findings suggest that upfront combinatorial inhibition of FGFR and MEK may represent a more effective treatment strategy for FGFR-driven gastric cancers.

Background Mutations and fusions in Fibroblast Growth Factor Receptor 3 (FGFR3) occur in 10–20% of metastatic urothelial carcinomas and confer sensitivity to FGFR inhibitors. However, responses to these agents are often short-lived due to the development of acquired resistance. The objective of this study was to identify mechanisms of resistance to FGFR inhibitors in two previously uncharacterised bladder cancer cell lines harbouring FGFR3 fusions and assess rational combination therapies to enhance sensitivity to these agents. Methods Acquired resistance to FGFR inhibitors was generated in two FGFR3 fusion harbouring cell lines, SW780 (FGFR3-BAIAP2L1 fusion) and RT4 (FGFR3-TACC3 fusion), by long-term exposure to the FGFR inhibitor BGJ398. Changes in levels of receptor tyrosine kinases were assessed by phospho-RTK arrays and immunoblotting. Changes in cell viability and proliferation were assessed by the Cell-Titre Glo assay and by propidium iodide staining and FACS analysis. Results Long term treatment of FGFR3-fusion harbouring SW780 and RT4 bladder cancer cell lines with the FGFR inhibitor BGJ398 resulted in the establishment of resistant clones. These clones were cross-resistant to the clinically approved FGFR inhibitor erdafitinib and the covalently binding irreversible FGFR inhibitor TAS-120, but remained sensitive to the MEK inhibitor trametinib, indicating resistance is mediated by alternate activation of MAPK signalling. The FGFR inhibitor-resistant SW780 and RT4 lines displayed increased expression of pERBB3, and strikingly, combination treatment with an FGFR inhibitor and the ATP-competitive pan-ERBB inhibitor AZD8931 overcame this resistance. Notably, rapid induction of pERBB3 and reactivation of pERK also occurred in parental FGFR3 fusion-driven lines within 24 h of FGFR inhibitor treatment, and combination treatment with an FGFR inhibitor and AZD8931 delayed the reactivation of pERBB3 and pERK and synergistically inhibited cell proliferation. Conclusions We demonstrate that increased expression of pERBB3 is a key mechanism of adaptive resistance to FGFR inhibitors in FGFR3-fusion driven bladder cancers, and that this also occurs rapidly following FGFR inhibitor treatment. Our findings demonstrate that resistance can be overcome by combination treatment with a pan-ERBB inhibitor and suggest that upfront combination treatment with FGFR and pan-ERBB inhibitors warrants further investigation for FGFR3-fusion harbouring bladder cancers.

The EGFR/RAS/MEK/ERK signaling pathway (ERK/MAPK) is hyper-activated in most colorectal cancers (CRCs). A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in CRC cells. Nevertheless, these drugs do induce expression of pro-apoptotic factors, suggesting they may prime CRC cells to undergo apoptosis. As histone deacetylase inhibitors (HDACi) induce expression of multiple pro-apoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger CRC cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in CRC cell lines and patient-derived tumor organoids in vitro,and attenuated Apc-initiated adenoma formation in vivo. Mechanistically, combined MAPK/HDAC inhibition enhanced expression of the BH3-only pro-apoptotic proteins BIM and BMF, and their knockdown significantly attenuated MAPK/HDAC inhibitor-induced apoptosis. Importantly, we demonstrate that the paradigm of combined MAPK/HDAC inhibitor treatment to induce apoptosis can be tailored to specific MAPK genotypes in CRCs, by combining a HDAC inhibitor with either an EGFR, KRASG12C or BRAFV600 inhibitor in KRAS/BRAFWT ; KRASG12C , BRAFV600E CRC cell lines respectively. These findings identify a series of ERK/MAPK genotype tailored treatment strategies that can readily undergo clinical testing for the treatment of colorectal cancer. Citation Format: Laura J. Jenkins, Ian Y. Luk, Walter D. Fairlie, Erinna F. Lee, Michelle Palmieri, Kael L. Schoffer, Tao Tan, Irvin Ng, Natalia Vukelic, Sharon Tran, Janson Tse, Rebecca Nightingale, Zakia Alam, Fiona Chionh, George Iatropoulos, Matthias Ernst, Shoukat Afshar-Sterle, Jayesh Desai, Peter Gibbs, Oliver M. Sieber, Amardeep S. Dhillon, Niall C. Tebbutt, John M. Mariadason. Genotype-tailored ERK/MAPK pathway and HDAC inhibition rewires the apoptotic rheostat to trigger colorectal cancer cell death [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B014.

Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf−/−) or specifically in the gut epithelium. Ehf−/− mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf−/− mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf−/−mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.

Despite having one of the lowest survival rates of all cancers, there have been no new approved treatments for malignant pleural mesothelioma (MPM) in over a decade. Standard-of-care treatment relies on Cisplatin plus Pemetrexed chemotherapy. Here, we tested a suite of BH3-mimetic drugs targeting BCL-2 pro-survival proteins of the intrinsic apoptotic pathway. We found BCL-XL is the dominant pro-survival protein in a panel of cell lines in vitro, though potent, synergistic cell killing occurred with MCL-1 co-targeting. This correlates with high-level expression of BCL-XL and MCL-1 in cell lines and a large cohort of patient tumour samples. BCL-XL inhibition combined with Cisplatin also enhanced cell killing. In vivo BCL-XL inhibition was as effective as Cisplatin, and the combination enhanced tumour growth control and survival. Genetic ablation of MCL-1 also enhanced the effects of BCL-XL inhibitors, in vivo. Combined, these data provide a compelling rationale for the clinical investigation of BH3-mimetics targeting BCL-XL in MPM.

BackgroundEts homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) transcription factors. EHF is specifically expressed in epithelial tissues, however its role in development and epithelial homeostasis is largely uncharacterized.MethodsWe generated a novel mouse strain in which the Ets DNA binding domain (exon 8) of Ehf was flanked by loxP sites (EhfLox/Lox). To inactivate Ehf in the whole body, EhfLox/Lox mice were crossed to CMVCre mice, which were then bred out to generate germline Ehf null (Ehf−/−) mice. To inactivate Ehf specifically in the intestinal epithelium, EhfLox/Lox mice were bred to tamoxifen-inducible VillinCre-ERT2 mice. EhfLox/Lox mice were also crossed to tamoxifen-inducible Cdx2CreERT2; ApcLox/+ mice to determine the impact of Ehf deletion on Apc-initiated colon cancer development.ResultsTranscripts encoding the Ets binding domain of EHF were effectively deleted in all tissues in Ehf−/− mice. Ehf−/− mice were born at the expected Mendelian ratio, but showed reduced body weight gain and developed a series of pathologies during their lifespan that led the majority of Ehf−/− mice to reach an ethical endpoint within one year of age. Most prominent of these were the development of papillomas in the chin, and abscesses in the preputial glands (males) or vulvae (females) which showed evidence of Staphylococcus and Proteus infection. Consistent with the development of papillomas, the epidermis of Ehf−/− mice showed evidence of mild hyperplasia. A subset of Ehf−/− mice also developed cataracts and corneal ulcers. EHF is highly expressed in the colonic epithelium and Ehf−/− mice displayed increased susceptibility to dextran sodium sulphate-induced colitis. This phenotype was confirmed in intestinal-specific Ehf knockout mice, and histopathological analyses revealed reduced numbers of goblet cells and extensive transcriptional reprogramming in the colonic epithelium. Finally, colon-specific deletion of Ehf enhanced Apc-initiated adenoma development, unveiling a novel, tumour suppressive role for EHF in colorectal cancer.ConclusionThe Ets DNA-binding domain of EHF is essential for post-natal homeostasis of the epidermis and colonic epithelium, and functions as a tumour suppressor in the colon.

The EGFR/RAS/MEK/ERK signalling pathway (ERK/MAPK) is hyper-activated in most colorectal cancers (CRCs). A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in CRC cells. Nevertheless, these drugs do induce expression of pro-apoptotic factors, suggesting they may prime CRC cells to undergo apoptosis. As histone deacetylase inhibitors (HDACi) induce expression of multiple pro-apoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger CRC cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in CRC cell lines and patient-derived tumour organoids in vitro, and attenuated Apc-initiated adenoma formation in vivo. Mechanistically this effect was mediated through induction of the BH3-only pro-apoptotic proteins BIM and BMF. Importantly, we demonstrate that this treatment paradigm can be tailored to specific MAPK genotypes in CRCs, by combining HDACi with EGFR, KRASG12C or BRAFV600 inhibitors in KRAS/BRAFWT; KRASG12C, BRAFV600E CRC cell lines respectively. These findings identify a novel ERK/MAPK genotype-targeted treatment paradigm for colorectal cancer.

Despite having one of the lowest survival rates of all cancers, there have been no new approved treatments for malignant pleural mesothelioma (MPM) in over a decade. Standard-of-care treatment relies on Cisplatin plus Pemetrexed chemotherapy. Here, we tested a suite of BH3-mimetic drugs targeting BCL-2 pro-survival proteins of the intrinsic apoptotic pathway. We found BCL-XL is the dominant pro-survival protein in a panel of cell lines in vitro, though potent, synergistic cell killing occurred with MCL-1 co-targeting. This correlates with high-level expression of BCL-XL and MCL-1 in cell lines and a large cohort of patient tumour samples. BCL-XL inhibition combined with Cisplatin also enhanced cell killing. In vivo BCL-XL inhibition was as effective as Cisplatin, and the combination enhanced tumour growth control and survival. Genetic ablation of MCL-1 also enhanced the effects of BCL-XL inhibitors, in vivo. Combined, these data provide a compelling rationale for the clinical investigation of BH3-mimetics targeting BCL-XL in MPM.

Purpose: Histone deacetylase inhibitors (HDACi) are epigenome-targeting small molecules approved for the treatment of cutaneous T-cell lymphoma and multiple myeloma. They have also demonstrated clinical activity in acute myelogenous leukemia, non–small cell lung cancer, and estrogen receptor–positive breast cancer, and trials are underway assessing their activity in combination regimens including immunotherapy. However, there is currently no clear strategy to reliably predict HDACi sensitivity. In colon cancer cells, apoptotic sensitivity to HDACi is associated with transcriptional induction of multiple immediate-early (IE) genes. Here, we examined whether this transcriptional response predicts HDACi sensitivity across tumor type and investigated the mechanism by which it triggers apoptosis. Experimental Design: Fifty cancer cell lines from diverse tumor types were screened to establish the correlation between apoptotic sensitivity, induction of IE genes, and components of the intrinsic apoptotic pathway. Results: We show that sensitivity to HDACi across tumor types is predicted by induction of the IE genes FOS, JUN, and ATF3, but that only ATF3 is required for HDACi-induced apoptosis. We further demonstrate that the proapoptotic function of ATF3 is mediated through direct transcriptional repression of the prosurvival factor BCL-XL (BCL2L1). These findings provided the rationale for dual inhibition of HDAC and BCL-XL, which we show strongly cooperate to overcome inherent resistance to HDACi across diverse tumor cell types. Conclusions: These findings explain the heterogeneous responses of tumor cells to HDACi-induced apoptosis and suggest a framework for predicting response and expanding their therapeutic use in multiple cancer types. Clin Cancer Res; 23(18); 5573–84. ©2017 AACR.

Summary Biliary tract cancers (BTCs) currently have no approved targeted therapies. Although genomic profiling of primary BTCs has identified multiple potential drug targets, accurate models are needed for their evaluation. Genomic profiling of 22 BTC cell lines revealed they harbor similar mutational signatures, recurrently mutated genes, and genomic alterations to primary tumors. Transcriptomic profiling identified two major subtypes, enriched for epithelial and mesenchymal genes, which were also evident in patient-derived organoids and primary tumors. Interrogating these models revealed multiple mechanisms of MAPK signaling activation in BTC, including co-occurrence of low-activity BRAF and MEK mutations with receptor tyrosine kinase overexpression. Finally, BTC cell lines with altered ERBB2 or FGFRs were exquisitely sensitive to specific targeted agents, whereas surprisingly, IDH1-mutant lines did not respond to IDH1 inhibitors in vitro. These findings establish BTC cell lines as robust models of primary disease, reveal specific molecular disease subsets, and highlight specific molecular vulnerabilities in these cancers., Graphical Abstract, Highlights • BTC cell lines harbor similar genomic alterations to primary tumors • Transcriptomic profiling of BTC cell lines identified two molecular subtypes • MAPK signaling is activated in BTC via multiple mechanisms • BTC lines with deregulated ERBB2 or FGFRs respond to specific targeted therapies, Biological Sciences; Genetics; Genomics; Cancer

Colorectal cancers (CRCs) are characterized by global hypomethylation and promoter-specific DNA methylation. A subset of CRCs with extensive and co-ordinate patterns of promoter methylation has also been identified, termed the CpG-island methylator phenotype. Some genes methylated in CRC are established tumor suppressors; however, for the majority, direct roles in disease initiation or progression have not been established. Herein, we examine functional evidence of specific methylated genes contributing to CRC pathogenesis, focusing on components of commonly deregulated signaling pathways. We also review current knowledge of the mechanisms underpinning promoter methylation in CRC, including genetic events, altered transcription factor binding, and DNA damage. Finally, we summarize clinical trials of DNA methyltransferase inhibitors in CRC, and propose strategies for enhancing their efficacy.

Activating mutations in BRAF (BRAFV600E) occur in ~10% of colorectal cancers (CRCs) and drive tumorigenesis through constitutive activation of MAPK signaling. In metastatic CRC, BRAF mutations are associated with poorer prognosis and resistance to conventional therapies, necessitating an urgent need to develop new treatments for these patients. BRAF inhibitors such as vemurafenib and dabrafenib have significant clinical activity in BRAF-mutant melanoma, however BRAF-mutant CRCs are largely refractory to these agents, due at least in part to feedback-relief mediated reactivation of MAPK signaling or alternate signaling pathway activation. Strategies to enhance the activity of BRAF inhibitors in BRAF-mutant CRC are therefore needed. Consistent with clinical observations, treatment of a panel of BRAF-mutant melanoma and CRC cell lines with vemurafenib resulted in significantly increased apoptosis in melanoma cell lines compared to CRC cell lines, where effects were largely cytostatic. To determine the mechanisms for this differential response we interrogated vemurafenib-induced gene expression changes in the two tumor types, focusing on altered expression of components of the intrinsic apoptotic pathway. Vemurafenib induced a more pronounced increase in expression of the pro-apoptotic genes BIM, BMF and PUMA and suppression of pro-survival gene MCL1 in melanoma cells compared to CRC cells. These findings suggested that the extent to which expression of pro and anti-apoptotic genes are altered by vemurafenib in CRC cells may be insufficient to reach the threshold required for apoptosis initiation. We therefore postulated that BH3-mimetics may synergize with vemurafenib to induce apoptosis in BRAF-mutant CRC cells. Analysis of quantitative proteomic data of BRAF-mutant CRC cell lines revealed significantly higher basal expression of the pro-survival proteins Bcl-xL and MCL1 compared to BCL2 and BCLW, suggesting CRC cells may be particularly dependent on Bcl-xL and MCL1 for survival. Indeed, combination treatment of BRAF-mutant CRC cells with the Bcl-xL inhibitor A-1331852 significantly enhanced apoptosis in the majority of BRAF-mutant CRC lines. Comparatively, combination treatment of vemurafenib with the MCL1 inhibitor S63845 induced a modest increase in apoptosis, while combination treatment with the BCL2 inhibitor ABT-199 had no effect on apoptosis, consistent with the low levels of BCL2 expression in these lines. Finally, we investigated the effect of combination treatment of vemurafenib with inhibitors of both Bcl-xL and MCL1. The triple combination further enhanced apoptosis in 3/5 cell lines, suggesting these cell lines are likely dependent on both Bcl-xL and MCL1 for survival. Collectively, these findings demonstrate that combining BRAF-inhibitors with Bcl-xL and/or MCL1 inhibitors may represent a novel strategy for treating BRAF-mutant CRC. Citation Format: Laura J. Jenkins, Fiona Chionh, Ian Y. Luk, Erinna F. Lee, Amardeep S. Dhillon, Niall Tebbutt, Walter D. Fairlie, John M. Mariadason. BRAF inhibitors synergize with BH3 mimetics to induce apoptosis in BRAF mutant colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2492.

The five-year survival rate for patients with metastatic colorectal cancer is less than 15%, necessitating an urgent need to develop novel therapeutic strategies for this disease. Loss of differentiation is associated with worse overall survival in colorectal cancer, suggesting that strategies to re-induce differentiation may provide clinical benefit. Differentiation therapy is effective in the treatment of acute promyelocytic leukemia but whether this approach can be efficacious in treating colorectal cancer is unknown. Inhibitors of the MAPK signaling pathway including the MEK inhibitor Trametinib can induce markers of differentiation in colorectal cancer cells. Trametinib also induces expression of Cdx-2, a known driver of colonic cell differentiation. Similarly, histone deacetylase inhibitors (HDACi) induce markers of differentiation in multiple tumor cell lines, including colon cancer cells. The aim of this study was to assess the potential therapeutic benefit of combining MEK (Trametinib) and HDAC (Panobinostat) inhibitors to further promote differentiation in colorectal cancer cells, and to elucidate the mechanistic basis for this effect. Combination treatment of HT29 and T84 cells with Trametinib and Panobinostat significantly enhanced mRNA and protein expression of the differentiation markers Cadherin 17 (CDH17) and Keratin 20 (KRT20), compared to either agent alone. Trametinib also induced expression of Cdx-2, an effect that was enhanced upon combination treatment with Panobinostat. Notably, Cdx-2 knockdown attenuated CDH17 and KRT20 induction by the combination, establishing a direct role for Cdx-2 in differentiation induction by the Trametinib/Panobinostat combination. Concomitant with inducing differentiation, the drug combination also invoked more apoptosis than either agent alone. To validate these findings in vivo, HT29 cells grown as xenografts were treated with Trametinib and Panobinostat alone and in combination. Mice treated with the combination had significantly smaller tumours than mice treated with vehicle or either agent alone. Consistent with the in vitro findings, immunostaining of the tumour xenografts demonstrated a strong increase in CDH17, KRT20 and Cdx2 expression upon treatment with the drug combination. Collectively, this study highlights a drug combination strategy to re-induce differentiation for the treatment of colorectal cancers. Citation Format: Laura J. Jenkins, Ian Y. Luk, Janson W. Tse, Jennifer Mooi, Amardeep S. Dhillon, John M. Mariadason. Combining MEK and HDAC inhibitors as a therapeutic strategy to promote differentiation of colorectal 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 3089. doi:10.1158/1538-7445.AM2017-3089