Colorectal Carcinoma Xenograft Model Research Articles

Tocilizumab Exerts Anti-Tumor Effects on Colorectal Carcinoma Cell Xenografts Corresponding to Expression Levels of Interleukin-6 Receptor.

The use of tocilizumab against the interleukin-6 receptor (IL-6R) has been demonstrated as inhibiting the progression of diverse cancers in vitro and in vivo. Nonetheless, evidence regarding the anti-tumor effects of tocilizumab on human colorectal carcinoma (CRC) corresponding to IL-6R expression levels remains scarce. To investigate the influence of IL-6R expression, SW480 and HT-29 cells inoculated subcutaneously into NU/NU mice were used as human CRC xenograft models with anti-IL-6R antibody (tocilizumab) therapy. The IL-6R expression levels, histology of CRC growth/invasiveness, and tumor growth-related signaling pathway were estimated by H&E and immunohistochemical staining. SW480 tumor cells with higher IL-6R expression levels showed better responsiveness in tocilizumab therapy than in the treated HT-29 group. Likewise, therapeutic effects of tocilizumab on the proliferative ability with mitotic index and Ki-67 expressions, invasiveness with MMP-9 proteinase expressions, and ERK 1/2 and STAT3 signaling transduction in the SW480 treatment group were superior to the HT-29 treatment group. In light of our results, IL-6R is the key indicator for the efficacy of tocilizumab treatment in CRC xenografts. From the perspective of precision medicine, tumor response to anti-IL-6R antibody therapy could be predicted on the basis of IL-6R expression levels. In this manner, tocilizumab may serve as a targeted and promising anti-CRC therapy.

Antitumor activities of a defucosylated anti‑EpCAM monoclonal antibody in colorectal carcinoma xenograft models.

Epithelial cell adhesion molecule (EpCAM) is a typeI transmembrane glycoprotein, which is highly expressed on tumor cells. As EpCAM plays a crucial role in cell adhesion, survival, proliferation, stemness, and tumorigenesis, it has been considered as a promising target for tumor diagnosis and therapy. Anti‑EpCAM monoclonal antibodies (mAbs) have been developed and have previously demonstrated promising outcomes in several clinical trials. An anti‑EpCAM mAb, EpMab‑37 (mouse IgG1, kappa) was previously developed by the authors, using the cell‑based immunization and screening method. In the present study, a defucosylated version of anti‑EpCAM mAb (EpMab‑37‑mG2a‑f) was generated to evaluate the antitumor activity against EpCAM‑positive cells. EpMab‑37‑mG2a‑f recognized EpCAM‑overexpressing CHO‑K1 (CHO/EpCAM) cells with a moderate binding‑affinity [dissociation constant (KD)=2.2x10‑8 M] using flow cytometry. EpMab‑37‑mG2a‑f exhibited potent antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/EpCAM cells by murine splenocytes and complements, respectively. Furthermore, the administration of EpMab‑37‑mG2a‑f significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab‑37‑mG2a‑f also exhibited a moderate binding‑affinity (KD=1.5x10‑8M) and high ADCC and CDC activities for a colorectal cancer cell line (Caco‑2 cells). The administration of EpMab‑37‑mG2a‑f to Caco‑2 tumor‑bearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab‑37‑mG2a‑f never suppressed the xenograft tumor growth of Caco‑2 cells in which EpCAM was knocked out. On the whole, these results indicate that EpMab‑37‑mG2a‑f may exert antitumor activities against EpCAM‑positive cancers and may thus be a promising therapeutic regimen for colorectal cancer.

Characterization of Anti-Angiogenic Chemo-Sensitization via Longitudinal Ultrasound Localization Microscopy in Colorectal Carcinoma Tumor Xenografts.

Objective:Super-resolution ultrasound localization microscopy (ULM) has unprecedented vascular resolution at clinically relevant imaging penetration depths. This technology can potentially screen for the transient microvascular changes that are thought to be critical to the synergistic effect(s) of combined chemotherapy-antiangiogenic agent regimens for cancer.Methods:In this paper, we apply this technology to a high-throughput colorectal carcinoma xenograft model treated with either the antiangiogenic agent sorafenib, FOLFOX-6 chemotherapy, a combination of the two treatments, or vehicle control.Results:Longitudinal ULM demonstrated morphological changes in the antiangiogenic treated cohorts, and evidence of vascular disruption caused by chemotherapy. Gold-standard histological measurements revealed reduced levels of hypoxia in the sorafenib treated cohort for both of the human cell lines tested (HCT-116 and HT-29). Therapy resistance was associated with an increase in tumor vascular fractal dimension as measured by a box-counting technique on ULM images.Conclusion:These results imply that the morphological changes evident on ULM signify a functional change in the tumor microvasculature, which may be indicative of chemo-sensitivity.Significance:ULM provides additional utility for tumor therapy response evaluation by offering a myriad of morphological and functional quantitative indices for gauging treatment effect(s).

The lncRNA DLGAP1-AS1/miR-149-5p/TGFB2 axis contributes to colorectal cancer progression and 5-FU resistance by regulating smad2 pathway

Colorectal carcinoma (CRC) ranks as the third most common malignancy. Long non-coding RNA DLGAP1-AS1 was reported to be dysregulated and to play a pivotal role in hepatocellular carcinoma (HCC). This work aims to analyze the functions and molecular basis of DLGAP1-AS1 in CRC progression and 5-fluorouracil resistance. Cell Counting Kit-8 (CCK-8) assay, Transwell assay, flow cytometry, and western blot were utilized to measure the CRC cell activity, invasiveness, and apoptosis. RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assay were adopted to verify the direct mutual action between DLGAP1-AS1 and miR-149-5p. The effect of DLGAP1-AS1 knockdown on tumor growth and chemosensitivity of 5-fluorouracil (5-FU) were investigated in the mouse CRC xenograft models. Functional assays showed that silencing DLGAP1-AS1 expression remarkably inhibited cell proliferation and aggressiveness ability and enhanced apoptosis rate and cell chemosensitivity to 5-FU. In addition, miR-149-5p was identified as a tumor suppressor and a direct downstream target of DLGAP1-AS1 in CRC. Furthermore, miR-149-5p was confirmed to directly bind to TGFB2 and DLGAP1-AS1 could regulate the expression of TGFB2 signaling pathway via miR-149-5p in CRC. These new findings indicate that DLGAP1-AS1 knockdown inhibited the progression of CRC and enhanced the 5-FU sensitivity of CRC cells through miR-149-5p/TGFB2 regulatory axis, suggesting that DLGAP1-AS1 may be a promising therapeutic target for CRC.

Resistance to anti-EGFR targeted therapy mediated by oncogenetic mutations in colorectal cancer: Revision of the dogma?

Traditionally, systemic treatment for high stage colorectal carcinoma (CRC) is mainly fluorouracil-based chemotherapy [1]. The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, by acting on specific molecular pathways in tumor growth or modulating immune response towards tumor cells, provide a more targeted response, a better side effect profile and greater impact on patient survival compared with conventional molecules. This monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor, is known to be effective only in a subset of KRAS wild-type colorectal cancers. Patients with mutations in either KRAS or NRAS gene are not eligible for anti-EGFR monoclonal antibody therapy [3]. This is due to downstream activation of the Ras/Raf/MAPK pathway by mutated RAS protein, leading to cell proliferation which cannot be sufficiently inhibited by anti-EGFR receptor monoclonal antibodies [4]. With the increasing choices of targeted agents, more and more biomarkers are tested. Currently, the standard recommended biomarker panel for colorectal carcinoma would include KRAS, NRAS, BRAF gene hotspot mutation detection and microsatellite instability test [5]. With the advances in genomic profiling and sequencing and the understanding of the resistance mechanisms, the contraindication of anti-EGFR therapy in mutant KRAS patients may be revised. Based on the fact that the KRAS mutation in CRC suppresses the phosphorylation of the AMP-activated protein kinase (AMPK) known to be toxic for the tumor cells, Hua et al obtained a satisfactory response to for the anti-EGFR antibodies in mutant KRAS CRC xenograft models after reactivation of the AMPK [6]. Knickelbein et al demonstrated that the anti-EGFR antibodies induce the death of CRC cells via a p73- dependent transcriptional activation of the pro-apoptotic Bcl-2 family protein (PUMA). This action is abolished in case of KRAS mutation. These authors admitted that the restoration of this pathway by inhibiting aurora kinases preferentially kills mutant KRAS CRC cells and overcomes KRAS-mediated resistance to anti-EGFR antibodies [7]. In fifty-one CRC patient-derived xenografts study, Lee et al showed that KRAS mutants expressed remarkably elevated autocrine levels of high-affinity EGFR ligands compared with wild-type KRAS. The use of an anti-EGFR IgG1 antibody that displays potent inhibitory effects on highaffinity EGFR ligand enhanced CRC KRAS mutant cells cytotoxicity [8]. Dealing with the resistance to targeted therapies in CRC patient looks feasible. It could allow to broaden the indications of anti-EGFR therapy and provide a better survival for a larger group of CRC patients. In this era of precision and personalized medicine, a complete case specific tumor profiling and the comprehensive study of the tumorogenesis mechanisms should allow to overcome the intrinsic and acquired resistance to these targeted high effective therapies.

Intratumoral exposure levels of pentaglutamated pemetrexed following treatment with LEAF-1401 and pemetrexed.

3524 Background: The activity of pemetrexed is highly dependent on the intracellular enzyme folypolyglutamate synthase (FPGS) which adds glutamates to pemetrexed and yields very potent pemetrexed polyglutamates. Pemetrexed pentaglutamate (tetraglutamated pemetrexed) is 80-fold more potent than pemetrexed in inhibiting thymidylate synthase. Yet it is a poor drug candidate because it cannot readily cross the negatively charged cell membrane due to its own negative charge. We are developing LEAF-1401, a novel nanoliposomal encapsulation of gamma L-pentaglutamated pemetrexed. Because liposomes can readily be taken up by tumor cells, for its anti-tumor effect, LEAF-1401 can directly deliver pentaglutamated pemetrexed into tumor cells, bypassing the need for transmembrane folate carriers and FPGS which are both downregulated in resistant tumors. Methods: To measure drug levels in tumor, blood and various tissues (biodistribution), in vivo testing of LEAF-1401 and pemetrexed was conducted in a CT-26 murine colorectal carcinoma xenograft model. Animals were treated with a single dose of either LEAF-1401 (80mg/kg; equivalent to 32 mg/kg pemetrexed) or pemetrexed (118mg/kg). Tumor growth inhibition and clinical assessments were conducted. Animals were sacrificed: 5 mice per timepoint in each group and tumor, blood, liver, spleen and other tissues were harvested. Pentaglutamated pemetrexed levels were quantitatively analyzed by LC/MS/MS. Results: Compared to pemetrexed, LEAF-1401 treatment resulted in a 19-fold increase in exposure levels of pentaglutamated pemetrexed in the tumor and significant tumor growth inhibition. Plasma levels of pentaglutamated pemetrexed were high with LEAF-1401, but undetectable with pemetrexed. Like other liposomes, LEAF-1401 also resulted in accumulation of pentaglutamated pemetrexed in the liver and spleen (See Table below). Treatment appeared to be generally well tolerated. Conclusions: LEAF-1401, given at approximately a quarter of the equivalent pemetrexed dose, resulted in a 19-fold increase in pentaglutamate pemetrexed in tumor tissue compared to regular pemetrexed. LEAF-1401 represents a promising new class of novel nanoliposomal antifolates, that enhance the intratumoral delivery of potent polyglutamate antifolates, and improve antitumor activity while retaining an acceptable safety profile. [Table: see text]

Efficacy of a Bispecific Antibody Co-Targeting VEGFA and Ang-2 in Combination with Chemotherapy in a Chemoresistant Colorectal Carcinoma Xenograft Model.

Vascular endothelial growth factor (VEGF) inhibition by the addition of bevacizumab to the chemotherapy regimen of metastatic colorectal cancer leads to an improved outcome. However, anti-angiogenic tumor therapy targeting a single factor may be limited by complementary mechanisms. Angiopoietin-2 (Ang-2, ANGPT2) is another important factor that cooperates with VEGF to drive tumor angiogenesis. It was shown that high Ang-2 levels are associated with a poor clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy. Therefore, combined inhibition of VEGF and Ang-2 was supposed to improve anti-angiogenic therapy. Here, we evaluated the efficacy of a bispecific antibody (CrossMab) co-targeting VEGF and Ang-2 in combination with chemotherapy in a chemoresistant colorectal carcinoma model. Antitumor activity was evaluated in athymic nude mice bearing subcutaneous DLD1 xenograft tumors and treated with anti-VEGF (B20), anti-Ang-2 (LC06) and anti-VEGF/Ang-2 (CrossMab) antibodies. Chemotherapy consisted of 5-FU and irinotecan. Resected tumors were analyzed immunohistochemically. First, an impact of targeting each single factor but also a clear advantage of co-targeting both factors could be demonstrated. Accordingly, tumor tissue showed strong staining for VEGF and Ang-2. Chemotherapy alone was less effective. Efficient tumor growth inhibition could be achieved by treatment with anti-VEGF/chemotherapy, single CrossMab and CrossMab/chemotherapy, which resulted in 3 out of 10, 6 out of 10 and 10 out of 10 complete responses, respectively, during seven weeks. Complete retarded tumors were characterized by massive intratumoral necrosis surrounded by layers of vital tumor cells and connective tissue with CD31-positive vessels at the periphery. In some cases, a distinct feature known as vessel co-option could be observed. In conclusion, the data from this model clearly support the strategy of co-targeting VEGF and Ang-2 and further demonstrate the beneficial impact of co-treatment with chemotherapy. The clear superiority of the CrossMab-containing regimen compared to clinical standard anti-VEGF/chemotherapy warrants further analyses in other models.

Abstract 3958: FAK inhibition reduces tumor infiltration of Tregs via restricting CCL5 release and boosts the present therapeutic regimen for BRAFV600E-mutated colorectal carcinoma

Abstract Background: BRAFV600E-mutated colorectal carcinoma (CRC) is a refractory disease with poor prognosis and limited therapeutic approaches. Recently, researches have indicated that the recruitment of Tregs in tumor tissue promoted by BRAFV600E is responsible for the weakened antitumor immunity. Whereas, as the main targeting therapy for this intractable carcinoma, BRAFV600E inhibitor has little response. In addition, FAK, being the novel therapeutic target in CRC, is considered to have an impact on the release of chemokines. In this study, we sought to explore the capacity of FAK inhibitor restricting Treg infiltration in tumor and its mechanism, as well as the synergistic antitumor effect with the present therapeutic strategy for BRAFV600E-mutated CRC. Methods: Cell proliferation was determined by CCK-8 assay and colony formation assay. Migration ability was detected by transwell assay. Cell cycle and cell group analysis were assessed by flow cytometry. Western blot, Elisa and RT-qPCR analysis were conducted for mechanism exploration. Immunohistochemistry and immunofluorescence analysis were performed to confirm the link between BRAFV600E, FAK and Tregs. CRC xenograft models were established to detect the Treg recruitment and evaluate the synergistic antitumor effect in vivo. Results: BRAFV600E-mutated CRC was of higher expression of FAK, and hence more sensitive to FAK inhibitor APG-2449. Besides, BRAFV600E-mutated in CRC was linked with more Treg infiltration in tumor site, therefore contributed to the attenuated antitumor immunity. What’s more, despite directly inhibiting tumor proliferation and migration, one of the important mechanisms of FAK blockade restricting the growth of BRAFV600E-mutated CRC, was by downregulating the release of Treg chemokine CCL5 from tumor cells through FAK-IL33 axis. With the recruitment of Tregs decreasing induced by FAK inhibitor APG-2449, the number of CD8+T cells relatively elevated in tumor tissue. In BRAFV600E-mutated CRC xenotransplantation models, we revealed that the combination of FAK inhibitor APG-2449 and Vemurafenib was of no significant difference with the combination of Vemurafenib and cetuximab. While adding APG-2449 to the current combination strategy achieved greater tumor suppressing effect than not adding. Conclusions: BRAFV600E-mutated CRC is associated with a higher expression level of FAK, as well as the tumor infiltration of Tregs. FAK inhibitor APG-2449 can promote antitumor immunity by restricting Treg number in tumor tissue, and shows a synergistic effect with the present therapeutic strategy for BRAFV600E-mutated CRC treatment. Citation Format: Luping Yuan, Wentao Pan, Suna Zhou, Qiuyun Luo, Xianglei Yan, Yuxin Zhang, Mengxian Pan, Lin Zhang, Miaozhen Qiu, Dajun Yang. FAK inhibition reduces tumor infiltration of Tregs via restricting CCL5 release and boosts the present therapeutic regimen for BRAFV600E-mutated colorectal carcinoma [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 3958.

FAK Inhibition Reduces Tumor Infiltration of Tregs via Restricting CCL5 Release and Boosts the Combination Therapy for BRAF V600E Mutant Colorectal Carcinoma

Background: BRAFV600E mutant colorectal carcinoma (CRC) is a refractory disease with poor prognosis and has little response to regular chemotherapy and targeted therapy. Recruitment of Tregs in tumor tissue promoted by BRAFV600E is responsible for the weakened antitumor immunity. FAK, a therapeutic target, has an impact on the release of Treg chemokines. Methods: Transwell assay, flow cytometry, western blot, Elisa and RT-qPCR analysis were conducted for mechanism exploration. Fluorescent multiplex immunohistochemistry analysis was performed to confirm the connection of BRAFV600E, FAK and Tregs. CRC xenograft models were established to detect the Treg recruitment and evaluate the synergistic antitumor effect in vivo. Results: CRC cells harboring BRAFV600E mutation was of higher expression of FAK, and hence more sensitive to the tumor inhibitory effect of FAK blockade. FAK inhibitor APG-2449 inhibited the proliferation via inducing G1 phase arrest and suppressed the migration of CRC cells, and APG-2449 reduced the tumor infiltrated Tregs through FAK-IL33-CCL5 axis. Overexpressed FAK in BRAFV600E mutant CRC led to larger amount of tumor infiltrated Tregs. BRAFV600E mutant CRC can benefit from FAK inhibition through downregulating Tregs in tumor site. Moreover, a synergistic antitumor effect of APG-2449 with Vemurafenib and/or Cetuximab in BRAFV600E mutant CRC was observed. Conclusions: BRAFV600E mutation in CRC is associated with higher expression level of FAK, and more Tregs infiltrated in tumors. FAK inhibitor APG-2449 has an impact on antitumor immunity of CRC by decreasing infiltrated Treg numbers, and boosts the targeted combination therapy of BRAF inhibitor and/or EGFR inhibitor for BRAFV600E mutant CRC. Funding Statement: This work was supported by National Natural Science Foundation of China (Grant number. 81602066 and 81772587); Guangdong Esophageal Cancer Institute Science and Technology Program (grant number M201809); CSCO-HengRui Oncology Research Fund (grant number Y-HR2018-184); the third outstanding young talents training plan and Medical Scientist program of Sun Yat-sen University cancer center; Science and Technology Planning Project of Guangzhou, China (Grant number. 201510161726583); National Science and Technology Major Project, China (Grant number. 2016ZX09101004); Natural Science Foundation of Guangdong Province, China (grant number 2018A030310260) and Medical Scientific Research Foundation of Guangdong Province, China (grant no. 2018102516469945). Declaration of Interests: The authors stated: No potential conflicts of interest were disclosed. Ethics Approval Statement: The animal experiments were approved by Animal Use and Care Committee of Sun Yat-sen University.

Antisense Oligonucleotides against miR-21 Inhibit the Growth and Metastasis of Colorectal Carcinoma via the DUSP8 Pathway

Accumulating research has documented that microRNA-21 (miR-21) plays an important role in the development of human colorectal carcinoma (CRC). Our recent work also showed that antisense oligonucleotides (ASOs) against miR-21 can impair the growth of CRC cells in vitro. However, the potential role of miR-21 in gene therapy against CRC remains to be fully elucidated. Here, we further observed the effect of ASOs against miR-21 on the growth and metastasis of CRC in vivo using a xenograft model of human CRC. We found that ASOs could effectively inhibit the growth and metastasis of CRC in vivo, accompanied by downregulated expression of miR-21 and reduced transduction of the AKT and ERK pathway. Mechanically, global gene expression analysis showed that the expression of DUSP8, a novel target of miR-21, was upregulated in tumor mass. Furthermore, overexpression of DUSP8 could remarkably suppress the proliferation and migration of CRC cells in vitro. Finally, downregulation of DUSP8 could abrogate the effects of ASOs against miR-21 on the proliferation and migration of CRC cells, as well as altered transduction of the AKT and ERK signaling pathway. Together, these data suggest that ASOs against miRNAs are an attractive and potential therapeutic for the treatment of human CRC and warrant further development.

Discovery of an Orally Bioavailable Dual PI3K/mTOR Inhibitor Based on Sulfonyl-Substituted Morpholinopyrimidines.

The discovery and optimization of a series of 2-morpholino-pyrimidine derivatives containing various sulfonyl side chains at the C4 position led to the identification of compound 26 as a potent dual PI3K/mTOR inhibitor. It exhibited high inhibitory activity against PI3Kα/β/γ/δ (IC50 = 20/376/204/46 nM) and mTOR (IC50 = 189 nM), potent functional suppression of AKT phosphorylation (IC50 = 196 nM), and excellent antiproliferative effects on a panel of cancer cells. Enzymic data and modeling simulation indicate that a cyclopropyl ring on the C4 sulfone chain and a fluorine on the C6 aminopyridyl moiety are responsible for its maintained PI3K activity and enhanced mTOR potency, respectively. Furthermore, compound 26 exhibited higher efficiency in the HT-29 colorectal carcinoma xenograft model at the daily dose of 3.75 and 7.5 mg/kg relative to BKM120 at the dose of 15 and 30 mg/kg.

Abstract DDT01-03: ABBV-621: A best-in-class TRAIL-receptor agonist fusion protein that enhances optimal clustering for the treatment of solid and hematologic tumors

Abstract Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of proteins that play diverse roles in the activation of several intracellular signaling pathways that control cell proliferation, survival, and apoptosis. Activation of the TRAIL pathway has been viewed as an attractive therapeutic approach for the treatment of cancer because of its distinct role in the induction of tumor cell apoptosis. TRAIL can bind as a trimer to membrane-bound or soluble receptors, and only the two closely related cell surface death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) can preferentially trigger the extrinsic pathway of tumor cells, leading to apoptotic cell death. The BCL-2 family member-driven mitochondrial apoptotic pathway can also serve to amplify the extrinsic pathway through further downstream activation of caspases. Given the crosstalk between the extrinsic and intrinsic cell death pathways, several resistance mechanisms have emerged to escape TRAIL-induced apoptosis. These include altered receptor regulation, overexpression of cellular FLICE-like inhibitory protein (cFLIP) that inhibits the proapoptotic activity of the DISC, overexpression of the prosurvival BCL-2 family member proteins, and the inhibitor of apoptosis proteins (IAPs) that directly inhibit caspases. In addition, TRAIL can induce non–cell death signaling pathways (e.g., NF-κB, MAPK, PI3K) to promote tumor cell growth as a potential resistance mechanism. Results of several clinical trials that targeted DR4 and DR5 with first-generation agonistic anti-TRAIL antibodies or human recombinant TRAIL indicated that these agents failed to demonstrate significant improvement in patient progression-free survival compared to standard-of-care therapy. The major contributing factor in this lack of clinical activity is believed to be the suboptimal TRAIL receptor clustering by these agonists. ABBV-621 is a novel, second-generation TRAIL receptor agonist comprising a human IgG1-Fc linked to native single-chain TRAIL-receptor binding domain (scTRAIL-RBD) monomers that are covalently connected by glycosylated linkers, resulting in two sets of trimeric RBDs. ABBV-621 is designed to maximize receptor clustering butdoes not require Fcγ-R-mediated crosslinking for optimal in vivo efficacy, which has been deemed an activity-limiting step for the competitor anti-DR4 and anti-DR5 antibodies in the clinic. In-house studies have demonstrated the requirement for enhanced caspase-8 aggregation into the death-inducing signaling complex (DISC) to induce potent tumor cell death, providing mechanistic insights as to why death receptor agonists originally failed and further distinguishing ABBV-621 from the first-generation agents. ABBV-621 induces dose-dependent apoptotic cell death at sub- to single-digit nanomolar potencies across a large panel (~100) of human hematologic and solid tumor cell lines in vitro. ABBV-621 activity is on-target and mechanism-based as demonstrated by a rapid activation of downstream apoptotic signaling events (cleavage of caspase-8, caspase-3, and PARP). In human tumor xenograft models, ABBV-621 exhibits potent antitumor activity in vivo as a monotherapy and in combination with targeted agents or chemotherapy using xenograft tumors derived from colorectal, lung, leukemia, and lymphoma cell lines. This activity is on-target, mechanism-based, and dose-dependent as indicated by rapid activation of tumor caspase-8 and caspase-3 after a single ascending dose of ABBV-621 in a colorectal carcinoma xenograft model. ABBV-621 exhibits a dose-proportional pharmacokinetic (PK) profile in mice and cynomolgus monkeys, and the projected human half-life is estimated to be ~2 days. PK/pharmacodynamic (PD) assessments estimate that achieving exposure above an efficacious concentration for 4-7 days is sufficient to maintain tumor regressions up to 4-7 weeks. Screens against 95 patient-derived xenograft (PDX) models consisting of 12 different solid tumor types uncovered single-agent activity (regressions) in >50% of all PDXs tested. PDX models can be more reflective of human disease by providing estimation of effects of tumor heterogeneity on ABBV-621 activity. Models that were partially sensitive or completely resistant to treatment were also identified, and collectively these responses are being utilized with associated pre- and post-treatment genomic/proteomic information to further inform potential patient stratification markers. To demonstrate on-target biologic activity in the clinic, proximal and distal target engagement markers will be pursued. In a GLP monkey toxicity study, ABBV-621 was well tolerated with no adverse test item–related findings. The first-in-human (FIH) phase 1/1b study with ABBV-621 will enroll subjects with previously treated advanced solid tumors and hematologic malignancies. The phase 1 objectives will be to establish the safety and tolerability of ABBV-621, as well as to understand the PK properties. Secondary objectives will be to explore target engagement and efficacy biomarkers and to evaluate clinical activity. Citation Format: Susan E. Morgan-Lappe. ABBV-621: A best-in-class TRAIL-receptor agonist fusion protein that enhances optimal clustering for the treatment of solid and hematologic tumors [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 DDT01-03. doi:10.1158/1538-7445.AM2017-DDT01-03

Structure-Based Optimization of Potent, Selective, and Orally Bioavailable CDK8 Inhibitors Discovered by High-Throughput Screening.

The mediator complex-associated cyclin dependent kinase CDK8 regulates β-catenin-dependent transcription following activation of WNT signaling. Multiple lines of evidence suggest CDK8 may act as an oncogene in the development of colorectal cancer. Here we describe the successful optimization of an imidazo-thiadiazole series of CDK8 inhibitors that was identified in a high-throughput screening campaign and further progressed by structure-based design. In several optimization cycles, we improved the microsomal stability, potency, and kinase selectivity. The initial imidazo-thiadiazole scaffold was replaced by a 3-methyl-1H-pyrazolo[3,4-b]-pyridine which resulted in compound 25 (MSC2530818) that displayed excellent kinase selectivity, biochemical and cellular potency, microsomal stability, and is orally bioavailable. Furthermore, we demonstrated modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8 activity, and tumor growth inhibition in an APC mutant SW620 human colorectal carcinoma xenograft model after oral administration. Compound 25 demonstrated suitable potency and selectivity to progress into preclinical in vivo efficacy and safety studies.

Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore.

Here we describe the discovery and optimization of 3-benzylindazoles as potent and selective inhibitors of CDK8, also modulating CDK19, discovered from a high-throughput screening (HTS) campaign sampling the Merck compound collection. The primary hits with strong HSP90 affinity were subsequently optimized to potent and selective CDK8 inhibitors which demonstrate inhibition of WNT pathway activity in cell-based assays. X-ray crystallographic data demonstrated that 3-benzylindazoles occupy the ATP binding site of CDK8 and adopt a Type I binding mode. Medicinal chemistry optimization successfully led to improved potency, physicochemical properties and oral pharmacokinetics. Modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8, was demonstrated in an APC-mutant SW620 human colorectal carcinoma xenograft model following oral administration.

Selective Allosteric Inhibition of MMP9 Is Efficacious in Preclinical Models of Ulcerative Colitis and Colorectal Cancer.

Expression of matrix metalloproteinase 9 (MMP9) is elevated in a variety of inflammatory and oncology indications, including ulcerative colitis and colorectal cancer. MMP9 is a downstream effector and an upstream mediator of pathways involved in growth and inflammation, and has long been viewed as a promising therapeutic target. However, previous efforts to target matrix metalloproteinases (MMPs), including MMP9, have utilized broad-spectrum or semi-selective inhibitors. While some of these drugs showed signs of efficacy in patients, all MMP-targeted inhibitors have been hampered by dose-limiting toxicity or insufficient clinical benefit, likely due to their lack of specificity. Here, we show that selective inhibition of MMP9 did not induce musculoskeletal syndrome (a characteristic toxicity of pan-MMP inhibitors) in a rat model, but did reduce disease severity in a dextran sodium sulfate-induced mouse model of ulcerative colitis. We also found that MMP9 inhibition decreased tumor growth and metastases incidence in a surgical orthotopic xenograft model of colorectal carcinoma, and that inhibition of either tumor- or stroma-derived MMP9 was sufficient to reduce primary tumor growth. Collectively, these data suggest that selective MMP9 inhibition is a promising therapeutic strategy for treatment of inflammatory and oncology indications in which MMP9 is upregulated and is associated with disease pathology, such as ulcerative colitis and colorectal cancer. In addition, we report the development of a potent and highly selective allosteric MMP9 inhibitor, the humanized monoclonal antibody GS-5745, which can be used to evaluate the therapeutic potential of MMP9 inhibition in patients.

Discovery of AM-7209, a potent and selective 4-amidobenzoic acid inhibitor of the MDM2-p53 interaction.

Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50 = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1.

Enhanced anti-colorectal cancer effects of carfilzomib combined with CPT-11 via downregulation of nuclear factor-κB in vitro and in vivo.

Upregulation of nuclear factor-κB (NF-κB) in colorectal carcinoma (CRC) accelerates tumor growth, whereas, irinotecan (CPT-11)-induced NF-κB activation reduces chemosensitivity and weakens the anti-colorectal cancer function itself, while proteasome inhibitors can inhibit NF-κB and improve the effect of chemotherapy. Carfilzomib (CFZ) is a novel proteasome inhibitor that has been recently approved by the FDA and is in clinical use for the treatment of multiple myeloma, but little is known about its activity against CRC. The aim of the present study was to explore whether CFZ alone or in combination with CPT-11 is effective in CRC treatment. We evaluated the novel therapeutic ability and mechanism of action of CFZ in CRC in vitro and in vivo. SW620 cells were incubated with CFZ alone or in combination with CPT-11. Cell proliferation was assessed by WST-1 and clonogenic assays, the cytotoxic interaction was assessed with a combination index (CI). Cell cycle progression was analysed with flow cytometry. Cell apoptosis was evaluated by detecting the Annexin V/propidium iodide (PI) ratio, caspase 3 and CD95 expression, and with TUNEL staining. Cell migration and invasion was determined with a wound-healing assay and a Transwell matrix penetration assay. A CRC xenograft model was established to monitor tumor growth. EMSA was used to analyse NF-κB activation and western blot analysis was used to detect the protein levels of related signaling factors. CFZ significantly inhibited the growth of SW620 cells, and had synergistic inhibitory effects with CPT-11 on survival and colony formation; possibly by inhibition of NF-κB activation, MEK/ERK and PI3K/AKT pathway factor dephosphorylation and survivin downregulation. Co-administration of CFZ and CPT-11 induced G2/M arrest, increased p21WAF1/CIP, and decreased mutant p53 and cdc25c expression. Induction of apoptosis was accompanied by marked increases in PARP cleavage, caspase 3 activation, an increase of CD95 and p-p38, and ATF3 activation. Combination treatment lowered the invasive and migration ability of SW620 cells, reduced MMP and increased TIMP protein expression. Finally, co-administration of CFZ and CPT-11 suppressed tumor growth and increased apoptosis compared with single-agent treatment in SW620 xenograft models correlated with NF-κB downregulation. Carfilzomib alone or in combination with CPT-11 is effective against colorectal cancer through inhibition of multiple mechanisms related to NF-κB, and could be a potential novel therapy for CRC.

Abstract C63: The Pelvic Radiation and Vorinostat (PRAVO) phase 1 study identifying MYC repression as biomarker of histone deacetylase inhibitor activity.

Abstract In modern radiation oncology, new insights into molecular radiobiology provide an opportunity for the rational integration of molecularly targeted therapeutics to optimize clinical radiation effects. One example is the use of histone deacetylase (HDAC) inhibitors as potentially radiosensitizing drugs. Conveyed by histone acetylation, HDAC inhibition causes perturbations in gene regulation implicated in cell cycle progression, DNA damage signaling and repair, and apoptosis. Following the demonstration that HDAC inhibitors enhanced radiation-induced clonogenic suppression in human colorectal carcinoma cell lines and xenograft models [1-3], the PRAVO study was conducted [4-5]. This trial, undertaken in patients treated with pelvic palliative radiotherapy (30 Gy in 3-Gy daily fractions) combined with the HDAC inhibitor vorinostat (administered once daily, three hours before radiation) for advanced gastrointestinal malignancy, was the first to report on the use of an HDAC inhibitor in clinical radiotherapy. It was designed to demonstrate that vorinostat reached the specific target (detection of tumor histone acetylation), the applicability of non-invasive tumor response assessment (using functional imaging), and importantly, that this combined-modality therapy was safe and tolerable. In the present report, potential biomarkers of vorinostat radiosensitizing action, not simultaneously manifesting molecular perturbations elicited by the radiation itself, were explored by gene expression array analysis of the PRAVO study patients’ peripheral blood mononuclear cells (PBMC), sampled at baseline (T0) and on-treatment two and 24 hours (T2 and T24) after the patients had received vorinostat. This strategy revealed 1,600 array probes that were common for the comparisons T2 versus T0 and T24 versus T2 across all of the patients, and furthermore, that no significantly differential expression was observed between the T0 and T24 groups. Functional annotation analysis of the array data showed that a significant number of the identified genes were implicated in biological processes and pathways comprising gene regulation (transcription, RNA processing), cell cycle progression (including p53 signaling, commonly involved in the DNA damage response), and chromatin biology. Of five genes that were selected both for verification of patients’ PBMC expression and for validation of vorinostat-regulated expression in human colorectal carcinoma xenograft models, transient repression of MYC was consistently observed in all conditions. In conclusion, within the design of the PRAVO study, all of the identified genes showed rapid and transient induction or repression and therefore, in principle, fulfilled the requirement of being pharmacodynamic biomarkers of vorinostat activity in fractionated radiotherapy, possibly underscoring the regulatory role of myc in this therapeutic setting. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C63. Citation Format: Anne Hansen Ree, Marie G. Saelen, Erta Kalanxhi, Ingrid H. G. Ostensen, Kristina Schee, Kathrine Roe, Torveig W. Abrahamsen, Svein Dueland, Kjersti Flatmark. The Pelvic Radiation and Vorinostat (PRAVO) phase 1 study identifying MYC repression as biomarker of histone deacetylase inhibitor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C63.

Chapter Ten - A Two-Hybrid Approach to Identify Inhibitors of the RAS–RAF Interaction

MCP compounds were developed with the idea to inhibit RAS/RAF interaction. They were identified by carrying out high-throughput screens of chemical compounds for their ability to inhibit RAS/RAF interaction in the yeast two-hybrid assay. A number of compounds including MCP1, MCP53, and MCP110 were identified as active compounds. Their inhibition of the RAS signaling was demonstrated by examining RAF and MEK activities, phosphorylation of ERK as well as characterizing their effects on events downstream of RAF. Direct evidence for the inhibition of RAS/RAF interaction was obtained by carrying out co-IP experiments. MCP compounds inhibit proliferation of a wide range of human cancer cell lines. Combination studies with other drugs showed that MCP compounds synergize with MAPK pathway inhibitors as well as with microtubule-targeting chemotherapeutics. In particular, a strong synergy with paclitaxel was observed. Efficacy to inhibit tumor formation was demonstrated using mouse xenograft models. Combination of MCP110 and paclitaxel was particularly effective in inhibiting tumor growth in a mouse xenograft model of colorectal carcinoma.

Radiosensitization by the histone deacetylase inhibitor vorinostat under hypoxia and with capecitabine in experimental colorectal carcinoma

BackgroundThe histone deacetylase inhibitor vorinostat is a candidate radiosensitizer in locally advanced rectal cancer (LARC). Radiosensitivity is critically influenced by hypoxia; hence, it is important to evaluate the efficacy of potential radiosensitizers under variable tissue oxygenation. Since fluoropyrimidine-based chemoradiotherapy (CRT) is the only clinically validated regimen in LARC, efficacy in combination with this established regimen should be assessed in preclinical models before a candidate drug enters clinical trials.MethodsRadiosensitization by vorinostat under hypoxia was studied in four colorectal carcinoma cell lines and in one colorectal carcinoma xenograft model by analysis of clonogenic survival and tumor growth delay, respectively. Radiosensitizing effects of vorinostat in combination with capecitabine were assessed by evaluation of tumor growth delay in two colorectal carcinoma xenografts models.ResultsUnder hypoxia, radiosensitization by vorinostat was demonstrated in vitro in terms of decreased clonogenicity and in vivo as inhibition of tumor growth. Adding vorinostat to capecitabine-based CRT increased radiosensitivity of xenografts in terms of inhibited tumor growth.ConclusionsVorinostat sensitized colorectal carcinoma cells to radiation under hypoxia in vitro and in vivo and improved therapeutic efficacy in combination with capecitabine-based CRT in vivo. The results encourage implementation of vorinostat into CRT in LARC trials.