Receptor Tyrosine Kinases VEGFR-2 Research Articles

Cabozantinib plus pembrolizumab as first-line therapy for cisplatin-ineligible advanced urothelial carcinoma (PemCab).

539 Background: Pembrolizumab (pem) was initially approved in the first line setting for cisplatin-ineligible metastatic urothelial carcinoma (mUC) patients (pts) with high PD-L1 expression and platinum ineligible pts regardless of PD-L1 status; this later changed to only platinum ineligible pts. There is a high disease progression (PD) rate with pem monotherapy. Cabozantinib (cabo) is a multiple receptor tyrosine kinase inhibitor targeting MET and VEGFR2 approved in multiple other malignancies as monotherapy or in combination with PD-1 inhibition. We hypothesized that combination of pem and cabo therapy will be safe and efficacious in patients with treatment naïve mUC. Methods: This is a phase II, open label, multi-center, single arm trial evaluating the tolerability and activity of pem 200 mg every 3 weeks and cabo 40 mg daily as first-line treatment for patients with mUC. Key inclusion criteria were histologically proven locally advanced or mUC, ECOG-PS 0-2, cisplatin-ineligible (including patient refusal of cisplatin), treatment naïve, and no prior PD-1/L1 therapy. The primary endpoint was objective response rate (ORR). Key secondary endpoints included progression-free survival (PFS) and overall survival (OS). There was a lead-in safety cohort of 6 pts. We hypothesized that combination therapy would improve ORR to > 32%. With 35 evaluable subjects, the lower bound of the 95% confidence interval would extend no more than 26% from the observed proportion. With ≥17 objective responses, the confidence interval excludes 32%. Results: Between December 26, 2018, and April 19, 2023, 36 pts were enrolled of which 35 were evaluable for response with the median follow up of 14.4 months (95% CI 12.2 – 16.2). The median age was 72.5y [range, 47-82]; ECOG PS 0-1 in 89% pts. Responses were observed in 15 pts (ORR= 42.8%) including complete responses in 5 pts (14.3%). Stable disease was seen in 10 pts (28.6%). The disease control rate was 68.5% (25/35). Median PFS was 7.7 months (95% CI 4.2 – 11.2). Fifty-two percent of pts developed any grade treatment-related adverse events (TRAE) attributable to either cabozantinib or pembrolizumab. The most common grade 1/2 TRAE were diarrhea, anorexia, dysgeusia, weight loss, and nausea. Forty-four percent pts developed grade 3/4 TRAEs most common being hypertension, hypophosphatemia, alanine transaminase elevation, diarrhea, and fatigue. Conclusions: This novel phase II trial of pem + cabo demonstrated a manageable toxicity profile and promising efficacy as first-line therapy in mUC including patient’s ineligible for cisplatin. Further investigation with a focus on predictive biomarkers is ongoing. Clinical trial information: NCT03534804 .

Discovery of acyl ureas as highly selective small molecule CSF1R kinase inhibitors

Based on the structure of an early lead identified in Deciphera’s proprietary compound collection of switch control kinase inhibitors and using a combination of medicinal chemistry guided structure activity relationships and structure-based drug design, a novel series of potent acyl urea-based CSF1R inhibitors was identified displaying high selectivity for CSF1R versus the other members of the Type III receptor tyrosine kinase (RTK) family members (KIT, PDGFR-α, PDGFR-β, and FLT3), VEGFR2 and MET. Based on in vitro biology, in vitro ADME and in vivo PK/PD studies, compound 10 was selected as an advanced lead for Deciphera’s CSF1R research program.

A phase II trial of pembrolizumab and cabozantinib in patients (pts) with recurrent metastatic head and neck squamous cell carcinoma (RMHNSCC).

6008 Background: Pembrolizumab (pembro) is an immune checkpoint inhibitor (ICI) approved for treating pts with recurrent/metastatic (RM) HNSCC. Cabozantinib (cabo) is a multiple receptor tyrosine kinase inhibitor (TKI) targeting MET and VEGFR2 shown to reduce tumor growth, metastasis, and angiogenesis and has immuno-modulatory properties. Methods: This is a phase II, open label multi-center, single arm trial evaluating the tolerability and clinical benefit of pembro administered at 200 mg every 3 weeks and cabo 40 mg daily for pts with RM HNSCC who have not received prior ICI. It had a lead-in safety cohort allowing reduction of cabo dose to 20mg daily. Eligible pts had RM HNSCC, deemed incurable, with a tumor PD-L1 CPS > 1, RECIST 1.1 measurable disease, a life expectancy of > 3 months, an Eastern Cooperative Group (ECOG) Performance Status (PS) of 0-1. Enrollment was initiated in March of 2019. We estimated that the ORR will improve to 35% with pembro+ cabo (from a historic of 18%) with a significance level of 0.05 and 80% power. Results: A total of 47 pts were screened, 13 screen failures [cavitation on scan (4), inability to swallow pills (2), other exclusions (7)]; 34 pts were enrolled, 32 were dosed and 31 evaluable (at least one follow-up scan). Pts had cancers of the oropharynx (21, 65%), nasopharynx (6, 19%), larynx/hypopharynx (4, 13%) and oral cavity (1,3%). 32 patients received cabo at 40 mg daily; 13 patients (41%) were dose reduced to 20mg daily [mucositis, increased liver function (LFT) tests, diarrhea]; males (n=29, 90%), median age 63 years (range 53-67); presence of distant metastases (34, 100%); prior radiation (29, 90%), chemotherapy (14, 43%); ECOG PS =0 (16, 50%), 1 (16,50%); HPV/p16 positive (17, 53%), negative (5, 16%), not applicable (10, 29%); the most frequent adverse event (AE) (all grades) was fatigue (16,50.0%), grade 3 or 4 treatment-related AE were increased LFTs, hyponatremia (3, 9.3% each). With a median follow up of 12.7 months (mos) (range 6.9- 20.5 mos), a RECIST 1.1 overall response rate ORR= 45.2% (CR=0; PR=14, 45.2%; SD =14, 45.2%; PD=3.0, 10%) with an overall clinical benefit of 90.4% were observed; The 1-yr OS was 67.7% (95% CI, 42.9%-83.6%; median 22.3 mos) and 1-yr PFS was 51.8% (95% CI, 28.8%-70.7%; median 14.6 mos). Conclusions: This phase II trial of pembro + cabo met its primary endpoint of ORR. The regimen is well-tolerated with very encouraging clinical activity in RM HNSCC and warrants further exploration in this disease. The study was supported by a grant from Exelixis to NFS. Clinical trial information: NCT03468218.

A phase 1 first-in-human study of XL092 administered alone or in combination with immune checkpoint inhibitors (ICIs) in patients (pts) with inoperable locally advanced or metastatic solid tumors: Description of genitourinary (GU) expansion cohorts.

TPS401 Background: XL092 is a novel oral multi-targeted inhibitor of receptor tyrosine kinases MET, VEGFR2, and TAM kinases (AXL, MER), which are important in tumor growth, neovascularization, and immune modulation of the tumor microenvironment. XL092 as a single-agent and in combination with an anti-PD-1 antibody showed antitumor activity in xenograft tumor models (Hsu et al. 2020). This first-in-human study is evaluating safety, pharmacokinetics (PK), and preliminary antitumor activity of XL092 with or without (w/wo) ICIs in pts with advanced solid tumors. Presented here are the trial design and details of GU cohorts. Methods: In this phase 1, multicenter, open-label, dose-escalation and expansion study (NCT03845166), pts will receive escalating doses of XL092 w/wo atezolizumab (ATEZO) 1200 mg Q3W or avelumab (AVE) 800 mg Q2W. The dose-escalation stage is enrolling pts with inoperable locally advanced or metastatic solid tumors using a 3+3 (single-agent) or rolling 6 (combination) design; the primary objective is to determine maximum tolerated dose and/or recommended dose of XL092 for further evaluation when administered w/wo ICIs; secondary objective is to evaluate plasma PK of XL092 and its metabolites alone and in combination with ICIs. Primary objectives for the expansion stage are to evaluate preliminary efficacy of XL092 w/wo ICIs by estimating objective response rate (ORR) by investigator (Inv) per RECIST 1.1 and for specific cohorts by estimating the % of pts with PFS at 6 months by Inv per RECIST 1.1; secondary objective is safety of XL092 w/wo ICIs via monitoring nonserious and serious adverse events (AEs) including immune related AEs and AEs of special interest. For GU cohorts in expansion stage, single-agent XL092 will be tested in: (i) previously treated advanced clear cell (cc) renal cell carcinoma (RCC; progression on 1-3 prior systemic regimens w/wo a sarcomatoid component); (ii) previously treated advanced non-cc RCC (progression on 1-3 prior systemic regimens); (iii) metastatic castration resistant prostate cancer (mCRPC; prior treatment with ≥1 novel hormonal therapy and taxane-based chemotherapy, ≤4 prior systemic regimens). XL092 + ATEZO will be tested in non-cc RCC and mCRPC cohorts. Pts with advanced urothelial carcinoma will be enrolled in 3 cohorts: (i) pts with ongoing CR/PR/SD after 4-6 cycles of first-line chemotherapy (gemcitabine + cisplatin and/or gemcitabine + carboplatin) will receive XL092 + AVE; (ii) pts with progression on/after prior ICI therapy (≤2 prior systemic therapies) will be randomized 1:1 to XL092 + AVE or single-agent XL092; (iii) pts with progression on/after first-line platinum-based chemotherapy will receive XL092 + AVE. The study is currently enrolling pts. Clinical trial information: NCT03845166.

IJU this issue

This issue contains 15 Original Articles, three of which are excellent prospective clinical trials, and one Urological Note. Cabozantinib is a multiple receptor tyrosine kinase inhibitor targeting MET (c-MET), VEGFR2, RET, AXL, KIT and TIE-2. In the phase III, randomized, open-label METEOR study, cabozantinib was associated with a significant progression-free survival benefit and a greater objective response rate versus everolimus in patients with metastatic renal cell carcinoma who had progressed after VEGFR-TKI. Tomita et al. (Niigata, Japan) carried out a bridging study to METEOR to evaluate the efficacy and safety of cabozantinib in Japanese patients. This phase II, open-label, single-arm study showed that cabozantinib provided clinical benefits in Japanese patients with renal cell carcinoma, with similar efficacy and safety to the non-Japanese population. Medical lasers are increasingly used to treat benign prostatic hyperplasia. Shoji et al. (Isehara, Japan) carried out a randomized prospective study to compare functional outcomes and complications over a period of 12 months in benign prostatic hyperplasia treated with thulium laser enucleation of the prostate or bipolar transurethral resection of the prostate. This study showed that thulium laser enucleation of the prostate is superior to bipolar transurethral resection of the prostate in resected volume, resected rates per prostate volume and transition zone volume, hemostasis, catheterization and hospitalization time, and decrease in prostate-specific antigen. However, excessive laser irradiation time in thulium laser enucleation of the prostate has the possibility to delay improvement of overactive bladder symptoms and decrease sexual function. Loose seeds and stranded seeds are available for low-dose rate brachytherapy. Each method has its own advantages and disadvantages, such as flexibility and fixation. Tanaka et al. (Kashihara, Japan) carried out a randomized controlled trial to compare the post-implant dosimetric parameters between the conventional method (using only the loose seeds) and the hybrid method. This study showed that the hybrid method of low-dose rate brachytherapy can achieve a higher coverage of the periprostatic region compared with the conventional method, while maintaining an acceptable level of urethral and rectal doses. The intravesical recurrence rate after transurethral resection of bladder tumor carried out for non-muscle-invasive bladder cancer (NMIBC) is high, and some risk factors for recurrence have been identified. Sazuka et al. (Chiba, Japan) evaluated the risk of the residual urine volume and preoperative pyuria in intravesical recurrence after transurethral resection of bladder tumor among NMIBC patients. They found that the presence of residual urine and pyuria raised the risk of intravesical recurrence after transurethral resection of bladder tumor for NMIBC. Furthermore, especially in men, the recurrence-free survival was significantly higher in patients without than with residual urine by propensity score matching analysis. Iwasawa et al. (Tokyo, Japan) investigated the relationship between fluid intake and tumor recurrence in NMIBC patients by using urine-specific gravity as an indicator of daily fluid intake. They found that high urine-specific gravity was associated with tumor recurrence, especially in patients not receiving adjuvant bacillus Calmette–Guérin therapy. This showed that increasing fluid intake might prevent recurrence in relatively low-risk NMIBC patients. Testicular microlithiasis (TM) is a relatively rare condition occurring in the testicular parenchyma that is characterized by multiple bright and small foci without acoustic shadow limited to the testicle on scrotal ultrasonography. TM is a characteristic of testicular dysgenesis syndrome and was shown to be associated with infertility. Hiramatsu et al. (Tokyo, Japan) evaluated the correlation between the degree of severity in TM and several factors. This study showed the prevalence of TM in normal healthy Japanese men who are trying for first-time pregnancy. This also showed that men with TM might have poor semen findings, and that men with severe TM might have even worse semen parameters. None declared.

33 Poster Discussion - XL092, a multi-targeted inhibitor of MET, VEGFR2, AXL and MER with an optimized pharmacokinetic profile

Background: Receptor tyrosine kinases (RTKs) MET, VEGFR2, AXL and MER are believed to play important roles in tumor growth, vascularization, resistance to therapy and in modulating the tumor immune microenvironment. Consistent with this the multi-targeted RTK inhibitor cabozantinib, a potent inhibitor of these RTKs as well as others, has shown significant clinical activity in a variety of solid tumors, and shows promising activity in combination with checkpoint inhibitors. Cabozantinib has an extended plasma half-life which results in drug accumulation during initial dosing. We sought to develop a novel RTK inhibitor that retains the target profile of cabozantinib but with a significantly shorter clinical half-life. The profile of XL092, a compound meeting these criteria, will be presented.

Targeting Receptor Tyrosine Kinase VEGFR-2 in Hepatocellular Cancer: Rational Design, Synthesis and Biological Evaluation of 1,2-Disubstituted Benzimidazoles.

In this study, a novel series of 1,2-disubstituted benzo[d]imidazoles was rationally designed as VEGFR-2 inhibitors targeting hepatocellular carcinoma. Our design strategy is two-fold; it aimed first at studying the effect of replacing the 5-methylfuryl moiety of the well-known antiangiogenic 2-furylbenzimidazoles with an isopropyl moiety on the VEGFR-2 inhibitory activity and the cytotoxic activity. Our second objective was to further optimize the structures of the benzimidazole derivatives through elongation of the side chains at their one-position for the design of more potent type II-like VEGFR-2 inhibitors. The designed 1,2-disubstituted benzimidazoles demonstrated potent cytotoxic activity against the HepG2 cell line, reaching IC50 = 1.98 μM in comparison to sorafenib (IC50 = 10.99 μM). In addition, the synthesized compounds revealed promising VEGFR-2 inhibitory activity in the HepG2 cell line, e.g., compounds 17a and 6 showed 82% and 80% inhibition, respectively, in comparison to sorafenib (% inhibition = 92%). Studying the effect of 17a on the HepG2 cell cycle demonstrated that 17a arrested the cell cycle at the G2/M phase and induced a dose-dependent apoptotic effect. Molecular docking studies of the synthesized 1,2-disubstituted benzimidazoles in the VEGFR-2 active site displayed their ability to accomplish the essential hydrogen bonding and hydrophobic interactions for optimum inhibitory activity.

New approaches in 3D modeling of in vitro growth of primary cultures of malignant gliomas

Background. The incidence of brain gliomas firmly occupies a leading position among all central nervous system tumors – 40–50 % of the cases detected, more than half of them are glioblastoma. Existing cell lines and cultivation methods do not reflect all the features of the three-dimensional (3D) organization of native glioblastoma. The use of temozolomide leads to the development of drug resistance and acute relapse, followed by a poor clinical outcome. The development of resistance is largely associated with the presence of tumor stem cells in the population and intratumoral heterogeneity. Obtaining 3D cultures from the primary material will allow us to save the stem cell pool and tumor-specific features.The study objective. Get a 3D model based on primary cell cultures, which allows you to save a heterogeneous population and the original phenotype of tumor cells.Materials and methods. We used U-87MG human glioma cells and GBM002 primary cell culture obtained from surgical material with a confirmed diagnosis of glioblastoma. Neurospheres were obtained from cell lines, the growth of which was monitored using the InCell Analyzer 6000 automatic cell analysis system. Flow cytometry was used to determine the CD133+ cell content. The expression of the receptor tyrosine kinases VEGFR1, VEGFR2 (endothelial growth factor type 1 and 2 receptors), FGFR2 (fibroblast growth factor receptor type 2) and the hypoxia marker HIF-1α (hypoxia inducible factor, 1α) in the neurospheres was evaluated using confocal microscopy.Results. GBM002 glioblastoma cells isolated from the surgical material formed neurospheres, while the number of CD133+ cells increased from 1–2 to 16–19 % compared with two-dimensional cultures. During long-term cultivation of cells with non-cytotoxic doses of temozolomide, it was found that such cells form smaller neurospheres compared to control cells. It was shown that the expression of receptor tyrosine kinases during cultivation of GBM002 glioblastoma cells in neurospheres differs from that in two-dimensional cultures. We found that in neurospheres, the expression of FGFR2 and VEGFR1, is significantly increased.Conclusion. 3D cultivation of primary cultures allows one to obtain a more heterogeneous population of tumor cells that reflects the spatial heterogeneity of cells, increase the pool of stem cells and recreate hypoxia conditions inside the brain micro-tumors.

Abstract C078: Substituted oxindoles as AMP-activated protein kinase (AMPK) inhibitors and their evaluation in models of leukemia

Abstract AMP-Activated protein kinase (AMPK) acts as a central metabolic sensor at the interface of metabolic and signaling networks. Activated AMPK promotes multiple catabolic processes to generate ATP, such as glucose uptake, glycolysis, fatty acid uptake and oxidation, and mitochondrial biogenesis. In addition, AMPK activation suppresses the cell cycle, and anabolic processes such as the mammalian Target of Rapamycin Complex 1 (mTORC1)-dependent protein synthesis and fatty acid biosynthesis via inactivating phosphorylation of cytosolic acetyl-CoA carboxylase 1 (ACC1) and mitochondrial ACC2. The activation of AMPK under conditions of energetic stress is modulated by an allosteric mechanism where the depletion of ATP promotes AMP binding at the γ subunit of AMPK with a subsequent conformational change in the catalytic α subunit. In the context of cancer AMPK has been reported to promote tumor survival and progression under hypoxia and the maintenance of cancer stem cells (CSCs). A major challenge to interrogating the therapeutic potential of targeting AMPK in cancer is the lack of potent and selective small molecule inhibitors. Compound C has been widely used as an AMPK inhibitor, but it lacks potency and has a poor selectivity profile. The multi-kinase inhibitor, sunitinib, has demonstrated potent nanomolar inhibition of AMPK activity, but has broad-spectrum activity across the kinome. Although sunitinib targets multiple kinases, its nanomolar potency for AMPK inhibition and large scope for chemical substitution on the core oxindole ring make it an attractive lead for AMPK inhibitor development. We have used a computational model of sunitinib docked into the ATP-binding site of the α subunit of AMPK to design and synthesize several series of oxindoles to examine structural modifications to improve AMPK inhibition and selectivity. These candidate inhibitors were evaluated against the activity of the α1 and α2 isoforms of AMPK using the TR-FRET assay and cell engagement was determined by ELISA, measuring p-ACC(Ser79), in the chronic myeloid leukemia K562 cell line. Our assays identified two novel, potent oxindole-based AMPK inhibitors that hadsignificantly reduced binding affinity when compared with sunitinib against members of the receptor tyrosine kinase (RTK) family, including VEGFR1, VEGFR2 and CSF1R, as well as BTK, a member of the B cell receptor (BCR) signaling pathway. Interestingly, cellular AMPK inhibition did not impact cell viability or result in cytotoxicity in K562 cells and may only sensitize these cells to chemotherapy. AMPK inhibition may be a more effective strategy to eliminate cancer cells in hypoxic microenvironments or against certain cell types, such as CSCs, that may be more reliant on AMPK for survival. Our current studies are designed to examine our AMPK inhibitors as single agents and in combination therapy in cancer cells grown under hypoxia and in CSC populations in order to eliminate these drug-resistant cancer cell populations with an aim to prevent cancer recurrence. In summary, we have developed two novel, potent AMPK inhibitors that were designed to interact with the DFG motif in the ATP-binding site of the α subunit of AMPK that are undergoing evaluation in advanced models of leukemia. Citation Format: Christopher J Matheson, Kimberly A Casalvieri, Donald S Backos, Craig T Jordan, Philip Reigan. Substituted oxindoles as AMP-activated protein kinase (AMPK) inhibitors and their evaluation in models of leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C078. doi:10.1158/1535-7163.TARG-19-C078

Virtual screening,docking and molecular dynamics simulation of selected phytochemical compounds bound to receptor tyrosine kinases:A correlative anti angiogenic study.

Screening of phytochemicals for their anti angiogenic potential has been a growing area of research in the current decade. The following study proposes virtual screening, drug likeliness and ADME filtering of specific phytochemical based compounds retrieved from "TIP - A Database of Taiwan Indigenous Plants". The study further subjects the filtered phytochemicals for their molecular docking analysis and molecular dynamics simulation studies against the prominent receptor tyrosine kinases EGFR, VEGFR-1 and VEGFR-2 involved in angiogenesis phenomenon. Among the various in silico analysis done and precise interpretations, the current study finally proposes 1- Hydroxycryprochine as one of the most potent lead in combating angiogenic phenomenon and thus cancer. The following study involves all such important use of in silico platforms, tools and analysis protocols which are expected to reproduce commendable results in wet lab studies. The proposed compound 1-hydroxycryprochine tends to justify its anti angogenic potential in all interactional and stability studies.

Abstract 4855: AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic receptor tyrosine kinases

Abstract Introduction: CD44v6, a member of the CD44 family, is an essential co-receptor for c-MET, RON and VEGFR-2. The receptor tyrosine kinases (RTKs) c-MET and RON, are key players in tumorigenic pathways, are activated by the ligands HGF and MSP, respectively and homo- or heterodimerize with each other or can be autoactivated due to amplification. The RTK VEGFR-2 is critical for angiogenesis in solid epithelial tumors. The peptidergic inhibitor AMC303, which allosterically and selectively binds to CD44v6, was investigated for its inhibitory effects on ligand-induced and autophosphorylated c-MET activation, and its impact on tumor growth and metastases in vivo. Methods: In vitro, tumor cells were incubated with AMC303 for 30 min prior to induction with an RTK-ligand. Analysis of RTK activation was carried out by Western blotting. For in vivo xenotransplantation human pancreatic tumor cells (L3.6pl) were orthotopically injected. Animals were treated with AMC303 i.p. at 0.1, 1, 10 mg/kg QOD or QWK for 3 weeks to address dose-dependency, the optimal treatment schedule was investigated with one, three or five times weekly administrations. Regression of metastases was investigated at 1 mg/kg QOD. Survival was evaluated with a dose of 1 mg/kg QOD. Immunohistochemical (IHC) analysis was performed for cleaved caspase 6, smooth muscle actin and CD31. Reduction of vessel permeability was assessed in Balb/C mice with the Miles assay and quantification of intra-tumoral FITC dextran after i.v. injection. Results: In vitro, AMC303 inhibited c-MET, VEGFR2 and RON phosphorylation induced by their respective ligands. Notably, AMC303 inhibited c-MET activation in cells with enhanced c-MET signaling caused by the exon 14 skipping mutation, but not c-MET auto-phosphorylation due to gene amplification. In vivo, AMC303 treatment attenuated primary xenograft tumor growth and metastasis and significantly prolonged survival. IHC on treated xenograft-tumors revealed an increase in apoptosis and necrosis along with a reduction in myofibroblast infiltration, angiogenesis and vessel permeability. Pharmacokinetic properties after i.p. administration of AMC303 revealed an terminal half-life of 6h. Administration schedules of AMC303 (1mg/kg) given one, three or five times per week had no significant impact on tumor growth attenuation. AMC303 treatment could be paused for 10 days without incurring significant tumor regrowth. Conclusions: AMC303 inhibits c-MET in vitro in a ligand-dependent fashion and in vivo significantly improved overall survival in an orthotopic pancreatic xenograft mouse tumor model. AMC303 effectively attenuated both primary and metastatic tumor growth by increasing apoptosis, reduction of angiogenesis and vascular normalization. These findings demonstrate the benefit of targeting multiple oncogenic RTK signaling pathways by AMC303. Citation Format: Tina Heumann, Vanessa Al-Rawi, Thorsten Läufer, Martin Augsten, Alexandra Matzke-Ogi, Oliver Coutelle. AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic receptor tyrosine kinases [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 4855.

Phase II study of sitravatinib in combination with nivolumab in patients with advanced or metastatic urothelial carcinoma (UC) after checkpoint inhibitor therapy (CIT).

TPS498 Background: Combination therapy with agents targeting molecular and cellular mechanisms of CIT resistance is a rational approach to restoring CIT efficacy in patients (pts) with immunotherapy-resistant UC. Sitravatinib is a tyrosine kinase inhibitor (TKI) that targets multiple closely-related receptor tyrosine kinase (RTK) pathways, including the split RTKs VEGFR-2 and KIT, as well as the TAM (TYRO3, AXL, and MER) RTKs. Inhibition of the split RTKs enhances antitumor activity by reduction of immunosuppressive regulatory T cells and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment (TME). Inhibition of TAM RTKs further restores a more immune-supportive TME by depletion of MDSCs and repolarization of tumor-associated macrophages to the M1 phenotype associated with secretion of pro-inflammatory cytokines. Given these pleiotropic immune-activating effects, the combination of sitravatinib with nivolumab is a rational approach to restoring or enhancing CIT clinical activity in pts with immunotherapy-resistant UC. This combination has been shown to be safe and tolerable in an ongoing Phase 2 study in pts with metastatic non-small cell lung cancer. Methods: This open-label Phase 2 study evaluates tolerability and clinical activity of sitravatinib in combination with nivolumab in pts with advanced or metastatic UC who experienced disease progression on or after CIT as the most recent systemic therapy. Enrollment is stratified by prior receipt of platinum-based chemotherapy. If the initial 2 cohorts are of high interest for efficacy, the protocol allows for addition of new cohorts to include pts previously treated with selected immunotherapies (anti-CTLA-4, anti-OX40 or anti–CD137 therapy) or who are CIT-naïve. The primary objective is Objective Response Rate. A Predictive Probability Design will be applied allowing for expansion to 40 pts per cohort. Sitravatinib is administered orally daily in continuous 28-day cycles at 120 mg; nivolumab intravenously at 240 mg every 2 weeks or 480 mg every 4 weeks. Status: The study is open for enrollment and recruitment is ongoing. Clinical trial information: NCT03606174.

Abstract 3960: The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes

Abstract Introduction: In cancer patients, the development of metastases is associated with a poor clinical prognosis. Metastatic dissemination requires cancer cells to detach from the primary tumor and invade adjacent or distant tissue. This process involves epithelial-to-mesenchymal transition (EMT), whereby tumor cells lose cell-cell adhesion mediated by E-cadherin repression. The CD44 family of transmembrane glycoproteins comprises alternatively spliced variants that are involved in many cellular processes. The isoform CD44v6 has been shown to play a major role in tumor growth and metastasis. CD44v6 acts as a co-receptor for the receptor tyrosine kinases (RTKs) c-MET, RON and VEGFR-2, and is preferentially expressed in a variety of epithelial cancers. We have previously shown that the co-receptor function of CD44v6 for c-MET is the driving force for metastasis. c-Met and RON facilitate metastasis through MAPK signaling and VEGFR-2 plays a role in angiogenesis. AMC303, an allosteric and selective peptide inhibitor of CD44v6, prevents phosphorylation of the CD44v6 dependent RTKs and respective cellular processes. Methods: The effect of AMC303 on cancer cell function in response to the growth factors HGF, MSP and VEGF was studied in various assays. Cell migration and invasion was analyzed in scratch assays and the Boyden Chamber. Morphological changes of EMT were monitored using microscopy in scattering assays in the presence of growth factors. EMT markers were analyzed by Western blot and immunohistochemistry. Tube formation was microscopically analyzed. Results: AMC303 dose dependently and at low nanomolar concentrations (IC50 10 - 20nM) prevents HGF- or VEGF-induced activation of c-MET/VEGFR-2 in various cancer cells of pancreatic, lung and colonic origin or endothelial cells, which cannot be overcome by excess HGF and VEGF. HGF- and MSP-dependent cell migration, invasion and wound closure in scratch assays was prevented by AMC303 using different cancer cell lines. AMC303 had a strong inhibitory effect on HGF- and MSP- induced cell scattering, a morphological feature of EMT. Consistent with this, AMC303 inhibited the downregulation of HGF induced EMT markers (E-cadherin). In primary endothelial cells, AMC303 blocked activation of VEGFR-2 and VEGF-A induced formation of a tubular network. Conclusions: Phosphorylation of CD44v6 dependent RTKs is inhibited in primary endothelial cells and different epithelial tumor cells by binding of AMC303 to the extracellular domaine of CD44v6. This novel mode of action results in strong anti-tumor and anti-metastatic effects of AMC303 involving inhibition of migration and invasion of tumor cells and prevention of EMT. AMC303 is currently tested in a First-in-Human Phase I/Ib study in cancer patients with solid epithelial tumors. Citation Format: Thorsten Läufer, Martin Augsten, Vanessa Al-Rawi, Birgit Hotz, Yvonne Heneka, Oliver Coutelle, Alexandra Matzke-Ogi. The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes [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 3960.

Expression of VEGFR-2 during Liver Regeneration after Partial Hepatectomy in a Bioluminescence Mouse Model

Background: Liver regeneration requires the formation of new blood vessels. Endothelial cell proliferation is stimulated by vascular endothelial growth factor (VEGF) and its receptor tyrosine kinase VEGFR-2. The aim of this study was to investigate VEGFR-2 expression in vivo during liver regeneration after partial hepatectomy (PHx). Methods: Transgenic VEGFR-2-luc mice were used in which the luciferase reporter gene was under control of the VEGFR-2 promoter. Following 2/3 PHx, the mice underwent in vivo bioluminescence imaging until the 14th postoperative day. Additionally, liver tissue was analyzed by immunohistochemistry, in vitro luminescence assays, and quantitative RT-PCR. Results: In vivo bioluminescence imaging showed a significant increase in VEGFR-2 promoter activity after PHx. Maximum signal was recorded on the 3rd day; 8 days postoperatively the signal intensity decreased significantly. On the 14th day, bioluminescence signal reached almost baseline levels. Immunohistochemistry, quantitative RT-PCR, and in vitro luminescence confirmed a significant increase on the 3rd day following resection. The mRNA expression of VEGFR-2 was significantly higher on day 3 than preoperatively as well as on day 8. Conclusion: In vivo bioluminescence imaging with transgenic VEGFR-2-luc mice is feasible and provides a convenient model for noninvasively studying VEGFR-2 expression during liver regeneration. This may facilitate further experiments with modulation of angiogenesis by different substances.

Abstract 4911: Allosteric inhibition of the Receptor Tyrosine Kinases c-MET, RON and VEGFR-2 via the co-receptor CD44v6 by the novel compound AMC303

Abstract Introduction: The receptor tyrosine kinases (RTKs) c-MET and RON and more importantly their crosstalk, play a crucial role in mediating local invasion, systemic dissemination and resistance in different types of cancer. Both RTKs are activated by the ligands HGF and MSP respectively and homo- or heterodimerize with each other. The RTK VEGFR-2 is the most important receptor for angiogenesis in solid epithelial tumors. CD44v6, a member of the CD44 family of transmembrane glycoproteins, was identified as an essential co-receptor for activation of c-MET, RON and VEGFR-2. AMC303, a peptide based allosteric and selective inhibitor of CD44v6, was investigated for its inhibitory effects on c-MET, RON and VEGFR-2 pathways in vitro and its effect on tumor growth and metastases in vivo. Methods: Affinity was determined using Microscale Thermophoresis. For in vitro blocking assays AMC303 was added 30 min prior to induction with the ligands. Analysis of RTK activation was carried out by western blot. Analysis of cell migration and invasion was performed with Boyden Chamber assays. VEGF-A induced tube formation was microscopically analyzed. For in vivo studies nude mice were orthotopically implanted with human tumor cells (L3.6pl). Animals were treated with AMC303 i.v. at 0.1, 1, 10 mg/kg QOD or QWK for 3 weeks. Regression of metastases was investigated at 1 mg/kg QOD. HPLC was used to detect the amount of AMC303 in the primary tumor lysates. Results: AMC303 binds to the ectodomain of CD44v6 with high affinity. In various pancreatic, breast, colon, lung and HNSCC tumor cell lines activation of c-MET and RON by their ligands was inhibited by AMC303 in vitro and consequently ligand induced cell scattering, migration and invasion was significantly reduced. In endothelial cells, activation of VEGFR-2 and VEGF-A induced formation of a tubular network was blocked by AMC303 treatment. In vivo treatment with AMC303 inhibited tumor growth in a dose dependent manner. The metastatic spreading of the primary tumor was prevented when animals were treated at early disease stage. Most strikingly, a marked regression of established liver metastases was observed at progressed disease state when animals were treated with AMC303 at 1 mg/kg QOD for 3 weeks. Conclusions: AMC303 inhibits activation of the RTKs c-MET and RON allosterically in different epithelial tumor cells and VEGFR-2 in endothelial cells by extracellular binding to CD44v6. This unique and novel mode of action results in a strong anti-tumor and anti-metastatic effect in vivo which together with its wide safety and tolerability window in preclinical toxicity studies is strongly supporting the clinical investigation. AMC303 is currently tested in a Phase I study in patients with solid epithelial tumors. Citation Format: Vanessa Al-Rawi, Thorsten Laeufer, Katrin Glocker, Yvonne Heneka, Alexandra Matzke-Ogi. Allosteric inhibition of the Receptor Tyrosine Kinases c-MET, RON and VEGFR-2 via the co-receptor CD44v6 by the novel compound AMC303 [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 4911. doi:10.1158/1538-7445.AM2017-4911

Discovery of novel substituted benzo-anellated 4-benzylamino pyrrolopyrimidines as dual EGFR and VEGFR2 inhibitors

The quinazoline scaffold is the main part of many marketed EGFR inhibitors. Resistance developments against those inhibitors enforced the search for novel structural lead compounds. We developed novel benzo-anellated 4-benzylamine pyrrolopyrimidines with varied substitution patterns at both the molecular scaffold and the attached residue in the 4-position. The structure-dependent affinities towards EGFR are discussed and first nanomolar derivatives have been identified. Docking studies were carried out for EGFR in order to explore the potential binding mode of the novel inhibitors. As the receptor tyrosine kinase VEGFR2 recently gained an increasing interest as an upregulated signaling kinase in many solid tumors and in tumor metastasis we determined the affinity of our compounds to inhibit VEGFR2. So we identified novel dually acting EGFR and VEGFR2 inhibitors for which first anticancer screening data are reported. Those data indicate a stronger antiproliferative effect of a VEGFR2 inhibition compared to the EGFR inhibition.

ANTITUMOR EFFECT OF SUNITINIB AND BORTEZOMIB ON BREAST CANCER CELL LINE IN VITRO

The discovery for effective combinations of anticancer drugs for treatment for breast cancer is the actual problem in the experimental chemotherapy. In this paper we conducted a study of antitumor effect of the combination of sunitinib and bortezomib against MDA-MB-231 and SKBR-3 breast cancer cell lines in vitro. We found that bortezomib in non-toxic concentrations can potentiate the antitumor activity of sunitinib. MDA-MB-231 cell line has showed great sensitivity to the combination of bortezomib and sunitinib in vitro. Bortezomib and sunitinib caused reduced expression of receptor tyrosine kinases VEGFR1, VEGFR2, PDGFRa, PDGFRß and c-Kit on HER2- and HER2+ breast cancer cell lines

Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors.

In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG) to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV) irradiation of the prodrugs original inhibitory activity was completely restored and even distinctly reinforced, as was the case for the prodrug 4. The presented results are a further evidence for caging technique being an interesting approach in the protein kinase field. It could enable spatial and temporal control for the inhibition of VEGFR-2. The described photoactivatable prodrugs might be highly useful as biological probes for studying the VEGFR-2 signal transduction.

VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis.

ABSTRACTVascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes.

The three receptor tyrosine kinases c-KIT, VEGFR2 and PDGFRα, closely spaced at 4q12, show increased protein expression in triple-negative breast cancer.

BackgroundTriple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer with poor prognosis and no targeted therapy available. Receptor tyrosine kinases (RTKs) are emerging targets in anticancer therapy and many RTK-inhibiting drugs are currently being developed. The aim of this study was to elucidate if there is a correlation between the protein expression of three RTKs c-KIT, VEGFR2 and PDGFRα, their gene copy number, and prognosis in TNBC compared to non-TNBC.MethodsTumor tissue samples from patients diagnosed with primary breast cancer were stained with immunohistochemistry (IHC) for protein assessment, and with fluorescence in situ hybridization (FISH) for gene copy number determination. Breast cancer mortality (BCM), measured from the date of surgery to death, was used as endpoint.ResultsThe cohort included 464 patients, out of which 34 (7.3%) had a TNBC. High expression of the three RTKs was more common in TNBC compared to non-TNBC: c-KIT 49% vs. 10% (P<0.001), PDGFRα 32% vs. 19% (P = 0.07) and VEGFR2 32% vs. 6% (P<0.001). The odds ratio (OR) of c-KIT, VEGFR2 and PDGFRα positivity, adjusted for tumor characteristics, was 6.8, 3.6 and 1.3 times higher for TNBC than for non-TNBC. 73.5% of the TNBC had high expression of at least one of the three investigated receptors, compared to 30.0% of the non-TNBC (P<0.001). Survival analysis showed no significant difference in BCM for TNBC patients with high vs. low c-KIT, PDGFRα or VEGFR2 protein expression. 193 (42%) tumors were evaluated with FISH. No correlation was seen between increased gene copy number and TNBC, or between increased gene copy number and high protein expression of the RTK.Conclusionc-KIT, VEGFR2 and PDGFRα show higher protein expression in TNBC compared to non-TNBC. Further investigation clarifying the importance of these RTKs in TNBC is encouraged, as they are possible targets for anticancer therapy.