BRAF Mutant Colon Cancers Research Articles

A single-arm, phase II study of autophagy modulation using hydroxychloroquine in addition to encorafenib and cetuximab or panitumumab in metastatic BRAF-mutated colorectal cancer refractory to standard therapy.

TPS262 Background: BRAF mutations drive 5-10% of colon cancers. BRAF + EGFR inhibition with encorafenib + cetuximab is standard second-line therapy, per the BEACON trial, but responses are short-lived. Autophagy induction is implicated as a mechanism for this acquired resistance to therapy. Preclinical models have shown that BRAF inhibition leads to increased ATF4 phosphorylation and an overreliance on oxidative phosphorylation, both markers of autophagy induction. Use of hydroxychloroquine (HCQ), an antimalarial agent with anti-autophagy activity, reduces such markers and improves the cellular metabolic profile. This was confirmed clinically in the BAMM trial, a phase I/II study in which patients with advanced BRAF-mutated melanoma received dabrafenib, trametinib and HCQ (Mehnert JM et al, Clin Cancer Res 2022). This study asks a similar question in advanced, BRAF-mutated colon cancer, of whether autophagy inhibition overcomes acquired resistance to BRAF inhibition. Methods: This is a Phase II, single-arm, open-label study to be conducted at Robert H. Lurie Cancer Center of Northwestern University, Chicago, IL. The study was approved by the Northwestern University Institutional Review Board on 9/22/22 (IRB #: STU00217727); clinicaltrials.gov listing is pending. Key eligibility criteria: BRAF-mutated, stage IV colon cancer, with at least one line of therapy; ECOG PS 0/1, preserved organ function, and measurable disease at baseline. Key exclusion criteria: prior treatment with BRAF/MEK inhibitors; history of acute/chronic pancreatitis, psoriasis, or porphyria. Patients will receive standard of care encorafenib 300 mg daily and cetuximab IV weekly (panitumumab IV q 2 weeks can be used per investigator discretion). After a 14-day lead-in, HCQ 400 mg BID will be added. Each cycle is 28 days. CT scans will be done every 2 cycles, and blood will be taken every 2 weeks during the first 2 cycles for metabolomics analysis. The trial utilizes a Simon 2-stage admissible design. 14 patients will be enrolled in stage 1; if 4 or more responses are observed, the trial will continue to Stage 2 in which an additional 24 patients will be enrolled for a total sample size of 38 patients. The primary endpoint is objective response rate (ORR). Secondary endpoints are safety/efficacy, progression-free survival, overall survival and duration of response/stable disease. The exploratory endpoint is changes in markers of autophagy over time. The null hypothesis is an ORR of 20% (per the BEACON trial), and the alternative hypothesis is an ORR of 40%. This is the first trial exploring autophagy modulation in advanced, BRAF-mutated colon cancer. This study aims to show that autophagy inhibition with HCQ circumvents acquired resistance to BRAF + EGFR inhibition in advanced colon cancer.

Abstract 1626: The orthotopic mouse study of cetuximab and dasatinib treatment on BRAF mutated colon cancer cells

Abstract Colorectal cancer is one of the most common cancer in the world. The mortality rate is the third among the top ten cancers. Among colorectal cancer patients, about 65-75% of patients have overexpression of epithelial growth factor receptors (EGFR). The antibody drug cetuximab that inhibits EGFR has also been developed. Moreover, overexpression of src protein is found in about 80% of patients with colorectal cancer. Higher levels of Src expression is related to the metastasis of colon cancer. Dasatinib is an oral taken tyrosine kinase inhibitor to inhibit the src phosphorylation. Therefore, in this study, the mechanism of cetuximab and dasatinib combined treatment in the orthotopic mouse model is investigated. The in vivo mechanism of EGFR and E-cadherin crosstalk in the mouse model is discussed. In this study, HT-29 colon cancer cells with BRAF V600E mutation were orthotopically injected into the cecum wall of BALB/c nude male mice. Two weeks later, cetuximab, dasatinib, and combined treatment of cetuximab and dasatinib were applied for three weeks. The tumor sizes were then recorded after sacrificed. Tumor Tissue were processed for tissue section with H&E staining. Immunohistochemistry (IHC) was applied to detect EGFR, p-Src, E-cadherin with specific antibodies. The protein expression levels were achieved by Western blot. In the results, the tumor size was smaller in cetuximab and dasatinib combined treated group. In IHC staining, the E-cadherin expression were not apparent in primary tumor site, however, were abundant in migrated cells. P-src expression was reduced in the dasatinib treated group and combined treatment group. In western blot result, the E-cadherin was increased after the treatment of dasatinib, and the p-src expression was inhibited after dasatinib treatment and combined treatment. Our results demonstrated cetuximab combined with dasatinib has the better result of suppression of BRAF mutated colon cancer cell growth and metastasis in the orthotopic mouse model. Citation Format: Yu-Cheng Weng, Yu-Lin Li, Xiang-Ling Hou, Hui Min Koo, Yi-Wen Lu, Viriya Adhiguna Winarso, Chia-Wen Lin, Wei-Ting Chao. The orthotopic mouse study of cetuximab and dasatinib treatment on BRAF mutated colon cancer cells [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 1626.

Prognostic Value of LINC-ROR (rs1942347) Variant in Patients with Colon Cancer Harboring BRAF Mutation: A Propensity Score-Matched Analysis.

Emerging studies show that long intergenic non-protein coding RNA, regulator of reprogramming (LINC-ROR) is aberrantly expressed in several types of cancer, including colon cancer (CC). LINC-ROR intronic variant rs1942347 may impact gene regulation and disease phenotype. We aimed to explore the potential association of LINC-ROR (rs1942347) with the clinicopathological features and outcome of CC cases. Archived FFPE (n = 180) CC samples were enrolled. Taq-Man allelic discrimination PCR was used for genotyping in propensity-matched cohorts with/without positive staining for mutant BRAF protein after eliminating confounders bias. The rs1942347*A allele variant was associated with high pathological grade, larger tumor size, distant metastasis, and mortality. Multiple logistic regression analysis adjusted by sex and BRAF mutation showed A/A genotype carriers to have 3 times more risk of early onset of cancer (OR = 3.13, 95%CI = 1.28–7.69, p = 0.034) than T/T genotype carriers. Overall analysis showed that rs1942347*A allele carriers had higher risk of mortality under heterozygote (OR = 2.13, 95%CI = 1.08–4.35, p = 0.003), homozygote (OR = 5.0, 95%CI = 1.69–14.29, p = 0.003), dominant (OR = 3.33, 95%CI = 1.20–9.09, p = 0.003), and recessive (OR = 2.63, 95%CI = 1.37–5.0, p = 0.011) models compared to T/T allele carriers. Stratified analysis by BRAF status revealed that the ancestor T/T allele conferred protection in BRAF mutant CC patients and was associated with a 73–93% reduced risk of mortality under heterozygote/homozygote comparison models. Using Kaplan–Meier curves, carriers of the A/A genotype had shorter survival times than T/T cohorts. The univariate Cox regression model revealed that the A/A genotype was associated with a 3.5 times greater mortality risk than the T/T genotype. However, after adjustment by multiple Cox regression analysis, the risk was insignificant. In conclusion, this is the first study identifying the potential association of the LINC-ROR (rs1942347) variant with CC prognosis.

The Sphingosine Kinase 2 Inhibitor ABC294640 Restores the Sensitivity of BRAFV600E Mutant Colon Cancer Cells to Vemurafenib by Reducing AKT-Mediated Expression of Nucleophosmin and Translationally-Controlled Tumour Protein.

Vemurafenib (PLX4032), small-molecule inhibitor of mutated BRAFV600E protein, has emerged as a potent anti-cancer agent against metastatic melanoma harboring BRAFV600E mutation. Unfortunately, the effect of PLX4032 in the treatment of metastatic BRAF mutated colorectal cancer (CRC) is less potent due to high incidence of fast-developing chemoresistance. It has been demonstrated that sphingolipids are important mediators of chemoresistance to various therapies in colon cancer. In this study, we will explore the role of major regulators of sphingolipid metabolism and signaling in the development of resistance to vemurafenib in BRAF mutant colon cancer cells. The obtained data revealed significantly increased expression levels of activated sphingosine kinases (SphK1 and SphK2) in resistant cells concomitant with increased abundance of sphingosine-1-phosphate (S1P) and its precursor sphingosine, which was accompanied by increased expression levels of the enzymes regulating the ceramide salvage pathway, namely ceramide synthases 2 and 6 and acid ceramidase, especially after the exposure to vemurafenib. Pharmacological inhibition of SphK1/SphK2 activities or modulation of ceramide metabolism by exogenous C6-ceramide enhanced the anti-proliferative effect of PLX4032 in resistant RKO cells in a synergistic manner. It is important to note that the inhibition of SphK2 by ABC294640 proved effective at restoring the sensitivity of resistant cells to vemurafenib at the largest number of combinations of sub-toxic drug concentrations with minimal cytotoxicity. Furthermore, the obtained findings revealed that enhanced anti-proliferative, anti-migratory, anti-clonogenic and pro-apoptotic effects of a combination treatment with ABC294640 and PLX4032 relative to either drug alone were accompanied by the inhibition of S1P-regulated AKT activity and concomitant abrogation of AKT-mediated cellular levels of nucleophosmin and translationally-controlled tumour protein. Collectively, our study suggests the possibility of using the combination of ABC294640 and PLX4032 as a novel therapeutic approach to combat vemurafenib resistance in BRAF mutant colon cancer, which warrants additional preclinical validation studies.

BRAF Mutation as a Potential Therapeutic Target for Checkpoint Inhibitors: A Comprehensive Analysis of Immune Microenvironment in BRAF Mutated Colon Cancer.

BRAF mutated colon cancer presents with poor survival, and the treatment strategies are controversial. The tumor microenvironment, which plays a key role in tumorigenesis as well as responses to treatments, of this subtype is largely unknown. In the present study, we analyzed the differences of immune microenvironments between BRAF mutated and BRAF wild-type colon cancer utilizing datasets from The Cancer Genome Atlas and Gene Expression Omnibus and confirmed the findings by tissue specimens of patients. We found that BRAF mutated colon cancer had more stromal cells, more immune cell infiltration, and lower tumor purity. Many immunotherapeutic targets, including PD-1, PD-L1, CTLA-4, LAG-3, and TIM-3, were highly expressed in BRAF mutated patients. BRAF mutation was also correlated with higher proportions of neutrophils and macrophages M1, and lower proportions of plasma cells, dendritic cells resting, and T cells CD4 naïve. In conclusion, our study demonstrates a different pattern of the immune microenvironment in BRAF mutated colon cancer and provides insights into the future use of checkpoint inhibitors in this subgroup of patients.

Abstract 1081: Genome-wide CRISPR screens reveal Hippo pathway activation as a resistance mechanism in BRAF mutant colon cancer

Abstract BRAF V600E mutations occur in a subset of colon cancers. These are typically resistant to chemotherapy and are associated with a poor outcome. Combination treatment with BRAF and EGFR inhibitors is superior to standard chemotherapy and has recently received FDA approval, however the early emergence of drug resistance is a significant clinical problem. Clinical studies of resistant patients have identified mutations and amplifications in the MAPK pathway as important resistance drivers. To identify novel non-MAPK dependent resistance mechanisms, genome-wide CRISPR/Cas9 knockout screens were performed to identify genes causing resistance to a combination BRAF/MEK/EGFR inhibitor regimen in the BRAF mutant HT29 and LS411N colon cancer cell lines. A number of strong resistance hits were identified but importantly, only 3 genes (CSK, ARID1A and STK11) were detected as significantly enriched in both cell lines screened. Two of these, CSK and ARID1A, have been shown to play a role in activation of the Hippo signaling pathway. The generation of CSK knockout colon cell lines confirmed resistance to BRAF/MEK/EGFR inhibition both in vitro and in vivo using xenograft models. Furthermore, since CSK is a negative regulator of SRC, re-sensitisation of resistant BRAF mutant CSK knockout cells was achieved by adding Src inhibitors (Dasatinib and Saracatinib) to the combination therapy. Nuclear localisation of the transcription factors YAP1 and WWTR1 and binding to TEAD family members are required for Hippo pathway activation and we confirmed significantly increased nuclear YAP1/WWTR1 in CSK knockout cells. Furthermore, YAP1/WWTR1 nuclear localisation in these cells was reversed by treatment with Src inhibitors. Novel pharmacological TEAD inhibitors have recently been developed and will also be used to confirm that resistance can be overcome by specifically targeting the Hippo pathway. Expression profiling of CSK knockout cells revealed significant enrichment of pathways associated with Hippo signaling. Genes involved in regulating Hippo pathway activation were also identified as CRISPR screen resistance hits in both lung and head and neck cancer cell lines, suggesting that this may be an important mechanism of resistance among other tumour types and not limited to the colon. Here we show that activation of the Hippo pathway is a potential MAPK-independent resistance mechanism in BRAF mutant colon cancer, readily reversible by rational pharmacological targeting. Given the development of specific Hippo pathway inhibitors and plans for their use in clinical trials, activation of Hippo signaling should be considered in resistant BRAF mutant colon cancer where alterations in the MAPK pathway are not detected. Citation Format: Jonathan S. Brammeld, Hannah Thorpe, Mar Arias Garcia, Stacey Price, Jamie Young, Matthias Pfeifer, Barbara Lupo, Kosuke Yusa, Livio Trusolino, Mathew Garnett, Andrea Bertotti, Chris Bakal, Ultan McDermott. Genome-wide CRISPR screens reveal Hippo pathway activation as a resistance mechanism in BRAF mutant colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1081.

Proteomic Profiling of BRAFV600E Mutant Colon Cancer Cells Reveals the Involvement of Nucleophosmin/c-Myc Axis in Modulating the Response and Resistance to BRAF Inhibition by Vemurafenib.

BRAFV600E mutations are found in approximately 10% of colorectal cancer patients and are associated with worse prognosis and poor outcomes with systemic therapies. The aim of this study was to identify novel druggable features of BRAFV600E-mutated colon cancer (CC) cells associated with the response and resistance to BRAFV600E inhibitor vemurafenib. Towards this aim, we carried out global proteomic profiling of BRAFV600E mutant vs. KRAS mutant/BRAF wild-type and double wild-type KRAS/BRAF CC cells followed by bioinformatics analyses. Validation of selected proteomic features was performed by immunohistochemistry and in silico using the TCGA database. We reveal an increased abundance and activity of nucleophosmin (NPM1) in BRAFV600E-mutated CC in vitro, in silico and in tumor tissues from colon adenocarcinoma patients and demonstrate the roles of NPM1 and its interaction partner c-Myc in conveying the resistance to vemurafenib. Pharmacological inhibition of NPM1 effectively restored the sensitivity of vemurafenib-resistant BRAF-mutated CC cells by down-regulating c-Myc expression and activity and consequently suppressing its transcriptional targets RanBP1 and phosphoserine phosphatase that regulate centrosome duplication and serine biosynthesis, respectively. Altogether, findings from this study suggest that the NPM1/c-Myc axis could represent a promising therapeutic target to thwart resistance to vemurafenib in BRAF-mutated CC.

AIM2 Inhibits BRAF-Mutant Colorectal Cancer Growth in a Caspase-1-Dependent Manner.

Absent in melanoma 2 (AIM2), a DNA sensor that plays an important role in natural immunity system, has been reported to participate in colorectal cancer (CRC) development. However, the functional role of AIM2 in BRAF-mutant CRC remains unclear. In this study, we first investigated AIM2 expression level in BRAF-mutant CRC tumor tissues. Overexpression of AIM2 in CRC cells was performed to investigate the effect of AIM2 on CRC cell viability, and cell death detection and caspase activity assay were performed to explore the mechanism that AIM2 impacts the growth of BRAF-mutant CRC cells. Moreover, we confirmed the antitumor effect of AIM2 in BRAF-mutant CRC cell-derived tumor xenograft (CDX) models as well as patient-derived organoids (PDOs). Herein, we reported that AIM2 expression was lower in BRAF-mutant than that in BRAF wild-type CRC tumor tissues. Restoring the expression of AIM2 in BRAF-mutant CRC cells greatly inhibits the tumor cell growth by inducing necrotic cell death. Mechanism studies revealed that AIM2-induced cell death is in a caspase-1-dependent manner. Additionally, overexpression of AIM2 significantly inhibits tumor growth and metastasis in BRAF-mutant CRC in vivo, which was further confirmed in BRAF-mutant CRC PDOs. Taken together, our data suggested that AIM2 inhibits BRAF-mutant colon cancer growth in a caspase-1-dependent manner, which may provide evidence to understand the pathogenesis of CRC with BRAF-mutant, as well as new strategies for manipulation of CRC.

A Triptolide Loaded HER2-Targeted Nano-Drug Delivery System Significantly Suppressed the Proliferation of HER2-Positive and BRAF Mutant Colon Cancer.

BackgroundColon cancer (CRC) was a malignant tumor and there were about 25% of patients with tumor metastasis at diagnosis stage. Chemotherapeutic agents for metastatic CRC patients were with great side effects and the clinical treatment results of advanced CRC were still not satisfactory. Human epidermal growth factor receptor 2 (HER2) is overexpressed in some CRC patients and is an effective target for CRC patient treatment. Anti-HER2 therapy had a beneficial role in the treatment of HER2-positive metastatic CRC with fewer side effects. CRC patients with BRAF mutations were resistant to HER2 antibodies treatment. Therefore, there was an urgent need to develop new therapeutic agents.MethodsHER2 targeted nanoparticles (TPLNP) drug delivery system loading triptolide (TPL) were prepared and identified. The effects of TPLNP and free TPL on cell viability, targeting and cell cycle progression on HT29 (BRAF mutation) with HER2 overexpression, were evaluated by Cell Counting Kit-8 (CCK8), Fluorescence Activating Cell Sorter (FACS) and immunofluorescence methods, respectively. The anti-tumor efficacies of TPLNP were evaluated in subcutaneous xenograft model of colon cancer and the survival rate, tumor volume, liver and kidney indexes of tumor-bearing mice were measured.ResultsTPLNP was small in nanosize (73.4±5.2nm) with narrow size distribution (PDI=0.15±0.02) and favorable zeta potential (pH=9.6, zeta potential: −57.3±6.69mV; pH=7.0, zeta potential: −28.7±5.1mV; pH=5.6, zeta potential: −21.1±4.73mV). Comparing with free TPL treatment group, TPLNP developed stranger colon cancer-killing efficiency in a dose- and time-dependent manner detected with CCK8 method; achieved good in vitro colon cancer targeting detected with flow cytometry and immunofluorescence experiments; enhanced more HT29-HER2 apoptosis and induced more cell cycle arrested in G1-S phase detected with FACS in vitro. As for in vivo antitumor response, TPLNP remarkably inhibited the growth of colon cancer in the colon cancer xenograft model, significantly improved the survival rate and did not exhibit significant liver and kidney toxicity in contrast with free TPL in vivo.ConclusionTPLNP was effectively against colon cancer with HER2 overexpression and BRAF mutation in pre-clinical models. In summary, the TPLNP appeared to be a promising treatment option for CRC in clinical application based on improved efficacy and the favorable safety profile.

34P Molecular markers of response to different chemotherapeutic agents in RAS / BRAF mutated colon cancer cell lines

34P Molecular markers of response to different chemotherapeutic agents in RAS / BRAF mutated colon cancer cell lines

Long term response on Regorafenib in non-V600E BRAF mutated colon cancer: a case report

BackgroundNon-V600E BRAF mutated colorectal cancer (CRC) is a rare disease entity with specific clinical features. These tumors are less likely to have microsatellite instability than CRC with a V600E BRAF mutation and often harbor a KRAS or NRAS mutation. Notably, median overall survival is longer than in wild-type BRAF CRC. Little is known about treatment possibilities in these patients.Case presentationWe present the case of a 59 year old patient with a rare mutation in BRAF codon 594, who progressed rapidly on all classical therapies but experienced a clear and long lasting response on treatment with Regorafenib.ConclusionLittle is known about therapies that can be effective in the rare non-V600E BRAF mutated CRCs. We present a patient who had a definite response to treatment with Regorafenib. There are no predictive markers that define a subset of CRC patients who benefit most from Regorafenib. The specific features of this non-V600E BRAF mutated CRC may be relevant in the exploration of predictive biomarkers for the efficacy of Regorafenib.

Sequence-dependent Combination of 5-Fluorouracil and Targeted Agents in RAF-mutant Colorectal Cancer Cell Lines

V-raf murine sarcoma viral oncogene homolog B (BRAF)-mutant colorectal cancer has limited therapeutic options for targeted agent and showed poor response to standard chemotherapy containing 5-fluorouracil (5-FU). We previously reported that 5-FU induced the expression of thymidine kinase 1 (TK1), a rate-limiting enzyme for thymidine salvage pathway, which is related with acquired resistance for 5-FU. As TK1 expression was dependent on growth-stimulatory signal such as Braf-MEK and PI3K-AKT pathway, we investigated whether the sequential treatment of 5-FU followed by targeted agent could induce synergy for cell viability and repress TK1 expression in compared with the treatment in reverse sequence. Treatment with 5-FU for 2 days followed by vemurafenib, a mutant BRAF inhibitor, for another 2 days induced significant increase of cell death in compared with the single treatment in colo205 and RKO cell lines. However, the combined treatment in reverse sequence showed the similar response to single treatment. Treatment of dactolisib (a PI3K/mTOR inhibitor) or MK-2206 (a AKT inhibitor) preceded by 5-FU induced cell death more than single agent. Induction of TK1 by 5-FU was abrogated by subsequent treatment with vemurafenib, dactolisib, or MK-2206, whereas 5-FU still induced TK1 expression in the cells pretreated with these targeted agents. In summary, the sequence-dependent treatment of 5-FU and targeted agents is effective through the extinction of 5-FU-induced TK1 activation, which could give a promising strategy for patients having BRAF-mutant colon cancers.

Schedule-dependent synergistic effects of 5-fluorouracil and selumetinib in KRAS or BRAF mutant colon cancer models

Combination of MEK inhibitor and 5-FU had showed limited efficacy in clinical trials. We previously reported that acquired resistance to 5-FU was related with continued activation of salvage pathway. Here we investigated whether combination of 5-FU and a MEK inhibitor had treatment sequence-dependent synergistic effects in KRAS or BRAF mutant colon cancer models. Treatment with 5-FU followed by selumetinib (FS) induced highest cell death and synergy compared with reverse (SF) and concomitant (cFS) treatment in six cell lines. SF or cFS combination induced synergy in 1 or 2 cell lines, respectively, in which the synergy was less than that by FS combination. FS enhanced apoptosis and decreased anchorage-independent growth. Induction of thymidine kinase 1, a rate-limiting enzyme in salvage pathway, by 5-FU was abrogated by subsequent treatment with selumetinib, and ERK reactivation after selumetinib was prohibited by pretreatment with 5-FU. FS altered mRNA expression in groups of genes distinct from SF. Administration of 5-FU (10 or 30 mg/kg/day) for 7 days, followed by selumetinib (10 or 25 mg/kg/day) for another 7 days, in colo205 and HCT8 xenograft models significantly decreased tumor growth compared with a single agent. However, co-administration in the reverse sequence did not show the difference in tumor size compared with the treatment of single agent. Decreased expression of Ki67 was observed in tumors from mice treated with FS. Our results suggest that sequential administration of 5-FU plus selumetinib would be a promising strategy for patients having KRAS or BRAF mutant colon cancers.

Pharmacological Inhibition of Casein Kinase 2 Enhances the Effectiveness of PI3K Inhibition in Colon Cancer Cells.

Serine/threonine kinase B-Raf proto-oncogene (BRAF) mutant colon cancer has a poor prognosis and there is an absence of targeted treatments for this subtype. Here, we investigated the effects of inhibition of casein kinase 2 (CK2) on the inhibitory effects of BRAF and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibition in BRAF-mutant colon cancer cells. Colon cancer cell lines with mutations in components of the mitogen-activated protein kinase (MAPK) and PI3K signalling pathway were used. Cell viability was determined after exposure to single agent and combinations of erlotinib (EGFR inhibitor), dabrafenib (MEK inhibitor), GDC0941 (PI3K inhibitor) and CX4945 (CK2 inhibitor). Western blots were used to examine MAPK and AKT serine/threonine kinase (AKT) pathway activation. Addition of CX4945 to dabrafenib did not enhance the antiproliferative effects of single-agent dabrafenib. Use of GDC0941 alone was highly effective in controlling growth of both BRAF-mutant and wild-type cells and this effect was enhanced by CK2 inhibition. Inhibition of the PI3K/AKT pathway is central to regulating growth of colon cancer cells and this can be enhanced by CK2 inhibition.

Abstract 1821: Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer

Abstract Background: Almost 10% of colon cancers harbor the canonical activating V600 BRAF mutation that in melanoma cancers has been shown to dramatically sensitize cells to targeted therapy and prolong survival. In the colon, such tumors are associated with worse survival. Recent clinical trials of combination therapies targeting the EGFR and MAPK pathways in these tumors have demonstrated impressive response rates. However, the clinical benefit has been limited by the rapid emergence of drug resistance. Identifying and targeting the genes complicit in this process and thus re-sensitize resistant cancer cells may be of clinical benefit. The landscape of drug resistance in cancer patients is composed of (a) loss-of-function (LoF), (b) gain-of-function (GoF) and (c) point mutation-mediated perturbations of genes, with often no single mechanism being predominant. Therefore, capturing the full breadth of resistance genes for any drug would require multiple genetic screens to be executed in parallel. Methods: We performed 3 genome-wide genetic screens in parallel in BRAF mutant colon cancer cells treated with a BRAF/MEK/EGFR inhibitor combination, to capture all of the resistance mechanisms described above. To screen for LoF events we used a genome-wide CRISPR/Cas9 sgRNA library. For GoF resistance events, a lentiviral-based insertional mutagenesis vector was used to randomly integrate the SFFV enhancer/promoter sequence throughout the genome. Finally, to saturate the genome of each cell line with point mutations we used N-Ethyl-N-nitrosourea (ENU) to randomly mutagenize every base in the genome. Each genetic screen was analysed separately to identify the relevant genes and pathways that confer drug resistance before aggregating the outputs of all three screens for a more comprehensive view of the drug resistance landscape. Results: The most recurrent and strongest hit from the CRISPR knockout screen was the inhibitor of Src family kinases, CSK. A Gaussian Kernal Convolution analysis for recurrent integrations detected in resistant clones following the insertional mutagenesis screen identified five genes as likely resistance candidates - BRAF, SOS1, MET, FRS2 and KRAS. Finally, Illumina exome sequencing of resistant clones following ENU mutagenesis revealed statistical enrichment for non-synonymous point mutations in three genes in the MAPK pathway, namely MAP2K1, NRAS and KRAS. Of note, loss of CSK was also shown to confer resistance to targeted therapies in other cancer types. Conclusion: Genome-wide genetic screens identified genes in the MAPK pathway likely to cause resistance clinically, and many of which are amenable to therapeutic targeting. Of note, the resistance as a result of loss of CSK instead activates Src signalling. It can be overcome through the addition of a Src inhibitor, and may be relevant across a range of cancer types as a resistance mechanism. Citation Format: Jonathan Brammeld, Marco Ranzani, Elizabeth A. Coker, Stacey Price, Theodoros I. Roumeliotis, Barbara Lupo, Mi Petljak, Steven P. Williams, Francesco Iorio, Francesco Sassi, Nanne Aben, Kosuke Yusa, Livio Trusolino, Lodewyk Wessels, David J. Adams, Mathew Garnett, Bissan Al-Lazikani, Jyoti S. Choudhary, Andrea Bertotti, Ultan McDermott. Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1821.

Abstract 3729: Developing cetuximab-staurosporine conjugate as the therapeutic medicine in KRAS/BRAF mutated colon cancer cells

Abstract Epidermal growth factor receptor (EGFR) is the key receptor for cancer cell growing, cetuximab is the monoclonal antibody that targets EGFR and was approved for treatment of colorectal, head and neck cancer. However, patients with KRAS /BRAF mutation are screened for the resistance. Kinases activities in the signal axes have been demonstrated the role in drug resistance in colon cancer cells. In this study, cetuximab was conjugated with kinases inhibitor staurosporine and the efficacy was examined in KRAS/BRAF mutated colon cancer cells. The cetuximab and staurosprone conjugate was prepared based on the amide bond formation or the thioether bond formation. Both cleavable and non-cleavable linkers were applied to the conjugate. KRAS and BRAF mutated colon cancer cell lines SW480 and HT-29 were treated with cetuximab, staurosporine, cetuximab and Staurosporine combination, cetuximab-staurosporine conjugate, cell viability and the molecular expression were monitored. In the result, staurosporine was demonstrated to suppress cetuximab induced Src activation which may contribute to drug resistance. Wild type cell (SW48) is sensitive (63% viability) to cetuximab treatment compared to BRAF and KRAS mutated cells. Cetuximab-staurosporine conjugate showed twice efficacy than cetuximab on wild type cell, and also resulted in greater toxicity (50% viability) in BRAF/KRAS mutated cells which are resistant to cetuximab treatment. Taken together, cetuximab-staurosporine conjugate has therapeutic potential in BRAF/KRAS mutated colon cancer cells. Citation Format: Wei-Ting Chao, Wei-Ting Sun, Hsiang-Ling Chiu, Wan-Chen Wei, Yi-Che Wu, Ling-Yi Kao, Yuan-Chiang Chung, Shih-Hsien Chuang. Developing cetuximab-staurosporine conjugate as the therapeutic medicine in KRAS/BRAF mutated colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3729.

Abstract 4035: Cetuximab combined with dasatinib reduces drug resistance in KRAS/BRAF mutated colorectal carcinoma cells

Abstract Colorectal carcinoma is the most common cancer worldwide. Cetuximab is the first-line target drug for late-stage metastatic colon cancer treatment; however, resistance to it was found in 45% patients having KRAS mutation. Therefore, the alternative signal axis to target mutated cancer cells is urgent and investigated. The role and the importance of Src has emerged in colon cancer; this study is to investigate the drug combined efficacy of cetuximab and dasatinib, the Src inhibitor and the underling molecular mechanism in KRAS/BRAF mutated cells. In this study, KRAS and BRAF mutated colon cancer cell lines SW480 and HT-29 were treated with cetuximab, dasatinib and fluorouracil (5-FU) solitary or combined. Cell viability and migration ability were monitored, and the molecular mechanisms were analyzed by Western blot for signal protein expressions. The in vivo study was carried with subcutaneous xenograft mouse model to evaluate the drug efficacy. The result showed that cetuximab treatment induced Src activation dose dependently, and induced matrix-dependent cell migration in SW480 cells. Cetuximab, dasatinib and 5-FU combined treatment compared to solitary treatment can reduce cell viability in either wild-type or KRAS/BRAF mutated colon cells. In the signal protein expressions, Src expression was suppressed sufficiently with dasatinib treatment; the phosphorylation of MAPK in SW480 cells and the phosphorylation of PI3K in HT-29 cells were downregulated after treated with cetuximab together with dasatinib and 5-FU. The drug combination was also demonstrated to suppress subcutaneous tumor growth as well. These findings demonstrate the combination of cetuximab, dasatinib and 5-FU can be the potential treating strategy for colon cancer cells with KRAS/BRAF mutation. Citation Format: Hsiang-Ling Chiu, Yi-Che Wu, Wan-Chen Wei, Ling-Yi Kao, Wei-Ting Chao. Cetuximab combined with dasatinib reduces drug resistance in KRAS/BRAF mutated colorectal carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4035.

Abstract 2173: Targeting CDK1 and MEK/ERK overcome mutant BRAF-mediated apoptosis resistance in human colorectal cancer cells

Abstract BRAFV600E mutation occurs in ~10% of human colorectal cancers (CRC) where it is associated with treatment resistance and poor prognosis. Data from TCGA and a clinical trial cohort identified a distinct subset of BRAF mutant colon cancers with deregulation of the cell cycle and overexpression of CDK1 (Barras D, et al, Clin Cancer Res. 2016). We tested the hypothesis that CDK1 inhibition can enhance apoptosis in BRAFV600E CRC cells. Since BRAF mutant cells show p-ERK activation, combined inhibition of CDK1 and MEK/ERK was evaluated. Using isogenic colon cancer cells, BRAF mutant alleles were shown to confer resistance to the CDK1 inhibitor, R0-3306, in a gene dose-dependent manner that was associated with reduced cleavage of caspase-3 and downstream PARP, and decreased pH2AX (DNA fragmentation marker). Ectopic BRAFV600E or constitutively active MEK mutant also conferred resistance to R0-3306. CDK1 siRNA was shown to increase cobimetinib-induced apoptosis, including annexin V labeling, and increased pH2AX. Furthermore, ERK siRNA or cobimetinib treatment attenuated CDK1 protein expression and increased R0-3306-induced pH2Ax. Moreover, treatment with R0-3306 plus cobimetinib significantly enhanced a caspase-dependent apoptosis and markedly reduced colony formation vs either drug alone in two CRC cell lines. Caspase-3 cleavage by R0-3306 + cobimetinib was dependent upon caspase-8. Mechanistically, CDK1 inhibition by R0-3306 suppressed the phosphorylation of procaspase-8 at Ser-387 shown by R0-3306 withdrawal which restored p-C8-S387 coincident with expression of the mitotic marker, pH3S10. In conclusion, inhibition of CDK1 can significantly augment apoptosis induction by MEK/ERK inhibition in BRAFV600E CRC cells, suggesting a novel therapeutic strategy to overcome mutant BRAF-mediated resistance. Apoptosis induction by CDK1 inhibitor (R0-3306) ± MEK/ERK inhibitor cobimetinibTreatmentTreatmentTreatementTreatmentCRC cell linesDMSOR0-3306 (5 µM)cobimetinib (5 µM)combinationAnnexin V+ (%)RKO13.43 ± 1.4132.55 ± 2.9315.04 ± 0.2652.72 ± 3.16Annexin V+ (%)HT-2911.22 ± 1.8442.44 ± 2.7025.29 ± 0.1665.62 ± 4.11 Citation Format: Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. Targeting CDK1 and MEK/ERK overcome mutant BRAF-mediated apoptosis resistance in human colorectal cancer cells [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 2173. doi:10.1158/1538-7445.AM2017-2173

Targeting of super-enhancers and mutant BRAF can suppress growth of BRAF-mutant colon cancer cells via repression of MAPK signaling pathway

Bromodomain and extra-terminal (BET) inhibitors suppress super-enhancers and show cytotoxicity against multiple types of tumors. However, early clinical trials with BET inhibitors showed severe hematopoietic toxicities, highlighting the need for sensitive tumors and rational combination strategies to enhance their therapeutic potential. Here, we identified colon cancer-specific super-enhancers that were associated with multiple oncogenic pathways, including the mitogen-activated protein kinase (MAPK) signaling pathway. Among the 14 colon cancer cell lines tested, their sensitivity to JQ1, a BET inhibitor, was not correlated with c-MYC expression. Three of four BRAFV600E-mutant cell lines were sensitive. Addition of JQ1 to vemurafenib, a specific mutant BRAF inhibitor, suppressed cell growth by arresting cell cycle progression and inducing apoptosis in the BRAFV600E-mutant cells. Mechanistically, the feedback activation of MAPK signaling pathway by vemurafenib was repressed by JQ1. Further, the addition of JQ1 to a BRAF inhibitor enhanced the in vivo anti-tumor effect. Thus, this study indicates the therapeutic potential of targeting of super-enhancers and mutant BRAF in patients with BRAFV600E-mutant colorectal cancer.

256 - Dual inhibition of BET and mutant BRAF in BRAF-mutant colon cancer cells suppresses oncogenic pathways and synergistically inhibits their growth

256 - Dual inhibition of BET and mutant BRAF in BRAF-mutant colon cancer cells suppresses oncogenic pathways and synergistically inhibits their growth