BRAFV600E Inhibitor Research Articles

Abstract A012: Emergence of MEK1 mutations in RAF or RAS-mutant solid tumors following treatment with a combination of selective type II RAF inhibitor belvarafenib and MEK inhibitor cobimetinib

Abstract Acquisition of ARAF mutations upon treatment with the potent and selective RAF dimer (type II) inhibitor belvarafenib has been previously observed, but whether these alterations or others are observed in patients treated with a combination of belvarafenib and the MEK inhibitor cobimetinib is unknown. The mutational profiles from circulating tumor DNA (ctDNA) were determined using FoundationOne liquid ctDNA assay from paired baseline (BL) and end of treatment (EOT) peripheral blood samples from 79 patients with locally advanced or metastatic RAF or RAS-mutant solid tumors treated with belvarafenib and cobimetinib in the HM-RAFI-103 trial (NCT03284502). The proportion of mutations were compared between BL and EOT using a McNemar’s test (unadjusted) to identify mutations significantly enriched or lost following treatment. ARAF mutations were not observed to significantly increase following treatment with belvarafenib + cobimetinib (4% BL vs 4% EOT in all solid tumors, p = 1; 6% BL vs 6% EOT in melanoma), and in patients with pre-existing ARAF mutations, the variant allele frequency did not consistently increase following treatment. The MEK1 gene had the greatest increase in mutations following treatment, present in 5% (4/79) patients at BL vs 16% (13/79) at EOT (p = 0.02) across all solid tumors, which included 44 colorectal cancer (CRC) cases (56%), and 18 melanoma (23%). While CRC comprised the largest fraction of all solid tumors in this study, it did not contribute the greatest gain in MEK1 mutations at EOT, with 7% (3/44) of CRC patients having MEK1 mutations at BL vs 14% (6/44) at EOT (p = 0.2). The indication with the greatest number of patients developing previously undetected MEK1 mutations at EOT was melanoma, where MEK1 mutations were present in 0% (0/18) patients at BL vs 28% (5/18) at EOT (p = 0.07). The MEK1 mutations gained in melanoma, which included Q58_E62del, P124L, and C121S, occurred in the N-terminal or catalytic core domains of MEK1, were known to be of functional impact, and all but one patient developing them had ≥ 1 known to be gain of function via the Clinical Knowledge Database; one patient gained three distinct MEK1 mutations following treatment. The MEK1 C121S mutation was previously observed to appear in a melanoma patient who developed resistance to the BRAF V600 inhibitor vemurafenib, and in vitro studies demonstrated it also conferred resistance to MEK inhibition (Wagle et al., 2011). Melanoma patients in the HM-RAFI-103 trial gaining MEK1 mutations at EOT were more commonly BRAF-mutant(mt) or NRAS non-mt melanoma at BL, but sample size is small and results were non-significant (p = 0.3 and 0.3, for association with BRAFmt and NRASmt, respectively, Fisher’s exact test). All melanoma patients who developed MEK1 mutations post treatment had either a partial response (1/5) or stable disease (4/5) as best overall response. Together, these data suggest the acquisition of gain of function MEK1 mutations following treatment of solid tumors, including melanoma, with combined type II RAF dimer and MEK inhibition. Citation Format: Stephanie Hilz, Marissa Chen, Malgorzata Nowicka, Harini Chakravarthy, Maryam Moshref, Luca Gerosa, Jennifer Eng-Wong, Cassie Chou, Young S. Noh, Yoon-hee Hong, Yibing Yan. Emergence of MEK1 mutations in RAF or RAS-mutant solid tumors following treatment with a combination of selective type II RAF inhibitor belvarafenib and MEK inhibitor cobimetinib [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A012.

Benzimidazole-Based Derivatives as Apoptotic Antiproliferative Agents: Design, Synthesis, Docking, and Mechanistic Studies.

A new class of benzimidazole-based derivatives (4a-j, 5, and 6) with potential dual inhibition of EGFR and BRAFV600E has been developed. The newly synthesized compounds were submitted for testing for antiproliferative activity against the NCI-60 cell line. All newly synthesized compounds 4a-j, 5, and 6 were selected for testing against a panel of sixty cancer cell lines at a single concentration of 10 µM. Some compounds tested demonstrated remarkable antiproliferative activity against the cell lines tested. Compounds 4c, 4e, and 4g were chosen for five-dose testing against 60 human tumor cell lines. Compound 4c demonstrated strong selectivity against the leukemia subpanel, with a selectivity ratio of 5.96 at the GI50 level. The most effective in vitro anti-cancer assay derivatives (4c, 4d, 4e, 4g, and 4h) were tested for EGFR and BRAFV600E inhibition as potential targets for antiproliferative action. The results revealed that compounds 4c and 4e have significant antiproliferative activity as dual EGFR/BRAFV600E inhibitors. Compounds 4c and 4e induced apoptosis by increasing caspase-3, caspase-8, and Bax levels while decreasing the anti-apoptotic Bcl2 protein. Moreover, molecular docking studies confirmed the potential of compounds 4c and 4e to act as dual EGFR/BRAFV600E inhibitors.

New pyrazolylindolin-2-one based coumarin derivatives as anti-melanoma agents: design, synthesis, dual BRAFV600E/VEGFR-2 inhibition, and computational studies.

Malignant melanoma is the most invasive skin cancer with the highest risk of death. The inhibition of BRAFV600E appears relevant for overcoming secondary resistance developed during melanoma treatment. BRAFV600E triggers angiogenesis via modification of the expression of angiogenic inducers, which play a crucial role in the metastasis of melanoma. Accordingly, the dual inhibition of the BRAFV600E/VEGFR-2 signaling pathway is considered a rational approach in the design of anti-melanoma candidates. In this study, a new class of pyrazolylindolin-2-one linked coumarin derivatives as dual BRAFV600E/VEGFR-2 inhibitors targeting A375 melanoma cells was designed. Target compounds were tailored to occupy the pockets of BRAFV600E and VEGFR-2. Most of the synthesized compounds demonstrated potent mean growth inhibitory activity against A375 cells. Compound 4j was the most active cytotoxic derivative, displaying an IC50 value at a low micromolar concentration of 0.96 μM with a significant safety profile. Moreover, 4j showed dual potent inhibitory activity against BRAFV600E and VEGFR-2 (IC50 = 1.033 and 0.64 μM, respectively) and was more active than the reference drug sorafenib. Furthermore, derivative 4j caused significant G0/G1 cell cycle arrest, induced apoptosis, and inhibited the migration of melanoma cells. Molecular docking showed that compound 4j achieved the highest ΔG value of -9.5 kcal mol-1 against BRAFV600E and significant ΔG of -8.47 kcal mol-1 against VEGFR-2. Furthermore, the structure-activity relationship study revealed that TPSA directly contributed to the anticancer activity of the tested compounds.

Design and synthesis of new 1,2,4-oxadiazole/quinazoline-4-one hybrids with antiproliferative activity as multitargeted inhibitors.

The combination of BRAF and tyrosine kinase (TK) inhibitors has been demonstrated to be highly effective in inhibiting tumor development and is an approach for overcoming resistance in clinical trials. Accordingly, a novel series of 1,2,4-oxadiazole/quinazoline-4-one hybrids was developed as antiproliferative multitargeted inhibitors. The structures of the newly synthesized compounds 9a-o were validated using IR, NMR, MS, and elemental techniques. 9a-o were tested as antiproliferative agents. The results showed that the majority of the tested compounds showed significant antiproliferative action with 9b, 9c, 9h, 9k, and 9l being the most potent. Compounds 9b, 9c, 9h, 9k, and 9l were tested as EGFR and BRAFV600E inhibitors. These in vitro tests revealed that compounds 9b, 9c, and 9h are strong antiproliferative agents that may act as dual EGFR/BRAFV600E inhibitors. 9b, 9c, and 9h were further investigated for their inhibitory effect on mutant EGFR (EGFRT790M), and the results showed that the tested compounds had considerable inhibitory action. Cell cycle study and apoptosis detection demonstrated that compound 9b exhibits cell cycle arrest at the G2/M transition. Molecular docking simulations reveal the binding mechanism of the most active antiproliferative agents.

New 1,2,3-triazole/1,2,4-triazole hybrids linked to oxime moiety as nitric oxide donor selective COX-2, aromatase, B-RAFV600E and EGFR inhibitors celecoxib analogs: design, synthesis, anti-inflammatory/anti-proliferative activities, apoptosis and molecular modeling study

A new series of bis-triazole 19a-l was synthesised for the purpose of being hybrid molecules with both anti-inflammatory and anti-cancer activities and assessed for cell cycle arrest, NO release. Compounds 19c, 19f, 19h, 19 l exhibited COX-2 selectivity indexes in the range of 18.48 to 49.38 compared to celecoxib S.I. = 21.10), inhibit MCF-7 with IC50 = 9–16 μM compared to tamoxifen (IC50 = 27.9 μM). and showed good inhibitory activity against HEP-3B with IC50 = 4.5–14 μM compared to sorafenib (IC50 = 3.5 μM) (HEP-3B). Moreover, derivatives 19e, 19j, 19k, 19 l inhibit HCT-116 with IC50 = 5.3–13.7 μM compared to 5-FU with IC50 = 4.8 μM (HCT-116). Compounds 19c, 19f, 19h, 19 l showed excellent inhibitory activity against A549 with IC50 = 3–4.5 μM compared to 5-FU with IC50 = 6 μM (A549). Compounds 19c, 19f, 19h, 19 l inhibit aromatase (IC50 of 22.40, 23.20, 22.70, 30.30 μM), EGFR (IC50 of 0.112, 0.205, 0.169 and 0.066 μM) and B-RAFV600E (IC50 of 0.09, 0.06, 0.07 and 0.05 μM).

Synthesis and Antiproliferative Potential of Thiazole and 4-Thiazolidinone Containing Motifs as Dual Inhibitors of EGFR and BRAFV600E.

Thiazole and thiazolidinone recur in a wide range of biologically active compounds that reach different targets within the context of tumors and represent a promising starting point to access potential candidates for treating metastatic cancer. Therefore, searching for new lead compounds that show the highest anticancer potency with the fewest adverse effects is a major drug-discovery challenge. Because the thiazole ring is present in dasatinib, which is currently used in anticancer therapy, it is important to highlight the ring. In this study, cycloalkylidenehydrazinecarbothioamides (cyclopentyl, cyclohexyl, cyclooctyl, dihydronapthalenylidene, flurine-9-ylidene, and indolinonyl) reacted with 2-bromoacetophenone and diethylacetylenedicarboxylate to yield thiazole and 4-thiazolidinone derivatives. The structure of the products was confirmed by using infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and single-crystal X-ray analyses. The antiproliferative activity of the newly synthesized compounds was evaluated. The most effective inhibitory compounds were further tested in vitro against both epidermal growth factor receptor (EGFR) and B-Raf proto-oncogene, serine/threonine kinase (BRAFV600E) targets. Additionally, molecular docking analysis examined how these molecules bind to the active sites of EGFR and BRAFV600E.

MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance

Metastatic melanoma poses significant challenges as a highly lethal disease. Despite the success of molecular targeting using BRAFV600E inhibitors (BRAFis) and immunotherapy, the emergence of early recurrence remains an issue and there is the need for novel therapeutic approaches. This study aimed at creating a targeted delivery system for the oncosuppressor microRNA 126 (miR126) and testing its effectiveness in combination with a phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) inhibitor for treating metastatic melanoma resistant to BRAFis. To achieve this, we synthesized chitosan nanoparticles containing a chemically modified miR126 sequence. These nanoparticles were further functionalized with an antibody specific to the chondroitin sulfate proteoglycan 4 (CSPG4) melanoma marker. After evaluation invitro, the efficacy of this treatment was evaluated through an invivo experiment using mice bearing resistant human melanoma. The co-administration of miR126 and the PI3K/AKT inhibitor in these experiments significantly reduced tumor growth and inhibited the formation of liver and lung metastases. These results provide evidence for a strategy to target an oncosuppressive nucleic acid sequence to tumor cells while simultaneously protecting it from plasma degradation. The system described in this study exhibits encouraging potential for the effective treatment of therapy-resistant metastatic melanoma while also presenting a prospective approach for other forms of cancer.

Personalized approach to anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma (ATC) represents the rarest but most aggressive tumor entity of the thyroid gland. In this respect, the treatment of advanced ATC has rapidly evolved in recent years. Recently, new personalized forms of treatment that address the somatic mutational status of the tumor have been increasingly used. The aim of this article is to provide an overview of current molecular-based and personalized treatment options for ATC. Acurrent literature search was performed with afocus on personalized molecular-based treatment options for ATC. The majority of patients suffering from ATC have an advanced tumor disease at the time of initial diagnosis. Despite multimodal treatment approaches consisting of surgery, external beam radiation therapy (EBRT) and chemotherapy (CTX), the prognosis of ATC is still poor. Accordingly, the focus of innovative treatment approaches is on molecular-based, individualized tumor therapy, including in particular BRAFV600E and multikinase inhibitors. The potential of the latter seems to lie particularly in combination therapy with immune checkpoint inhibitors. These treatment options can be used in both adjuvant and neoadjuvant settings. Neoadjuvant treatment of advanced ATC can achieve apotentially resectable treatment setting and improve the poor prognosis of affected patients; however, larger prospective and randomized studies on these combination therapies are currently pending. The focus of future treatment approaches for ATC will be on individualized, molecular-based tumor therapy. In particular, the neoadjuvant use of these therapies may change the paradigm of ATC surgery as locally advanced as well as metastatic carcinomas can be converted to apotentially resectable status and made amenable to surgery.

INNV-40. DURABLE 10-YEAR RESPONSE TO VEMURAFENIB IN A PATIENT WITH CNS HAIRY CELL LEUKEMIA

Abstract Hairy cell leukemia (HCL) is an uncommon form of chronic B-cell lymphoproliferative disease, accounting for approximately 2% of all cases of leukemias. Although there have been rare instances of HCL affecting the central nervous system (CNS), these cases usually involve highly fatal leptomeningeal disease or direct leukemic infiltration into the brain. The BRAF V600E mutation, leading to the constitutive activation of the MAPK/ERK signaling pathway, is recognized as a key activating mutation in HCL and is present in almost all HCL patients. First-line treatment for HCL remains purine analogs; however, up to 20% of patients only achieve a partial response. Vemurafenib, an oral inhibitor of BRAF V600E, has demonstrated strong anti-tumor activity in patients with systemic relapsed/refractory HCL and has good CNS penetration. In this case study, we present a 67-year-old patient with HCL who initially presented with multifocal cranial neuropathies In August of 2013. His brain MRIs demonstrated multiple enhancing leptomeningeal foci. While a combination of cytarabine, methotrexate, and rituximab initially prevented disease progression, he experienced multiple relapses over the course of 2 years and eventually became refractory to treatment. Subsequently, the patient was started on vemurafenib, which resulted in the complete resolution of all lesions observed on MRIs. This response has persisted for 10 years since starting the medication. We suggest that besides its high response rate in systemic relapsed/refractory HCL, vemurafenib may offer benefits for patients with CNS involvement who are refractory to conventional chemotherapies and can achieve a durable response. Further investigation is needed to potentially establish vemurafenib as a first-line treatment option for HCL patients with CNS involvement.

Targeted NGS Panel Guides Risk-Adapted Treatment Intention in Newly Diagnosed Myeloma Patients

Introduction While overall survival has improved, patients with high-risk multiple myeloma (MM) have poor outcomes. Fluorescent in situ hybridisation (FISH) is widely used to identify high-risk genomic features at diagnosis, but a significant minority of patients classified with standard-risk disease progress rapidly and in retrospect may have warranted more intense frontline therapy. Recently, we implemented the Myeloma Genome Project Panel (MGPP) into clinical workflows - a myeloma-specific targeted sequencing panel capturing 228 genes, 6 translocation regions and 56 copy number abnormalities (CNAs) in a single assay [PMID: 35522533]. Given that MGPP captures many prognostic regions beyond the limited number assessed by FISH, there is potential to better identify high-risk patients. However, the utility of MGPP in supporting clinical risk designation and therapy choice when compared with FISH in newly diagnosed MM is unknown. Methods A prospective cohort study was conducted to compare the impact of MGPP vs. FISH on baseline risk stratification and treatment intention, in 55 unselected newly diagnosed MM patients. For MGPP, libraries were generated from 100ng DNA from bone marrow (BM) derived CD138 + MM cells and matched germline peripheral blood (Roche KAPA HyperCap Target Enrichment/Hyperplus kit). Reads were aligned to hg38, and somatic mutations, CNAs and translocations called with a custom pipeline. ISO-accredited FISH analysis was performed in NHS laboratories. In a multicentre multidisciplinary team (MDT) meeting, four blinded haematologists independently reviewed each clinical case presented twice with either FISH or MGPP, in a random order. Clinicians designated risk status and treatment intentions using a structured proforma. Therapeutically actionable variants could be selected as per the MyDRUG trial [ClinicalTrials.gov: NCT03732703]. A consensus MDT outcome for high-risk or more intensive therapy required at least 3 out of 4 concordant decisions. Results Data is presented here for 23 of 55 patients. The mean age was 68.9 years, 57% were transplant eligible, with R-ISS-1 (35%), R-ISS-2 (26%) and R-ISS-3 (39%). Firstly, MGPP had greater technical success than FISH, reporting successfully in 100% vs. 83% cases from the same diagnostic BM samples respectively (n=4 assay failures with FISH). Secondly, MGPP reported more genomic features than FISH, detecting 231 vs. 18 somatic variants at 127 vs. 7 regions, respectively. Thirdly, MGPP captured equivalent loci at greater resolution, detecting subclonal CNAs missed by FISH (Figure 1). In a head-to-head comparison with FISH, these additional genomic insights from MGPP influenced clinical treatment decisions. A clinician-led MDT reclassified 9 (39%) patients designated as standard-risk by FISH to high-risk status when presented with MGPP (Table 1). Reclassified patients were enriched for prognostically significant MYC translocations (17%) and subclonal 17p loss (22%), 1p loss (17%) and 1q gain (13%) CNAs that were missed by FISH. Altered risk designation influenced frontline treatment intention; clinicians opted for intensification of induction therapy in 13% (FISH) vs. 48% (MGPP) high-risk patients, and considered tandem autologous transplant as an option in 0% (FISH) vs. 23% (MGPP) transplant-eligible patients. FISH identified a therapeutically actionable variant in 3 (13%) patients [venetoclax, n=3]. MGPP identified 13 (57%) additional patients with opportunity for targeted therapy [MEK inhibitor, n=11; BRAF V600E inhibitor, n=2], which the MDT considered as a treatment option in all cases if available in trial. The full dataset and analysis will be presented at time of the conference. Conclusions We demonstrate that MGPP impacts clinical risk designation and treatment intention in newly diagnosed MM. MGPP had greater technical assay success than FISH and captured a greater range and depth of somatic variants. Importantly, these additional insights led a clinical MDT to reclassify 39% patients as high-risk, consider intensification regimen in 39% cases and potential for actionable targeted therapy in 57% patients. Our results support MGPP as a robust alternative to FISH in supporting risk-adapted treatment decisions. We will implement MGPP in additional large prospective cohorts over the next year, to better understand its prognostic utility and feasibility in routine clinical workflows.

Responses to Combination Chemotherapy with MAPK Pathway Inhibition in Relapsed and Refractory LCH

MAPK pathway inhibition (MAPKi) is associated with high response rates in patients with Langerhans cell histiocytosis (LCH), but monotherapy may not be curative and efficacy in LCH-associated neurodegeneration (LCH-ND) is not known. Based on upregulation of Bcl-xL by MAPK activation, we hypothesize that MAPKi may render LCH cells more sensitive to chemotherapy-induced cytotoxicity. We assessed the safety and efficacy of combination therapy in 10 patients with refractory systemic disease and LCH-ND. Records of patients treated with combination therapy at Texas Children's Hospital from October 2018 to July 2023 were reviewed for clinical responses as well as peripheral blood/tissue BRAFV600E mutation status, ataxia rating scores, and toxicity. The median age at the start of combination therapy was 6.82 years (range, 0.48-20.45 years) with a median of 4 treatments prior to this therapy regimen (range, 1-14). Almost all Clofarabine was administered outpatient (typical starting dose 25 mg/m2/day x 5 days). MAPK inhibitors included BRAFV600E inhibitors (50%) and MEK inhibitors (50%). Overall response was 8/10 (80%), with 5/5 (100%) objective response rate (ORR) for systemic disease. Further, 5/6 (83%) demonstrated reduced or extinguished peripheral BRAFV600E+ fraction by the end of combination therapy. Ataxia rating scores were improved in 2/3 (67%) individuals with clinical neurodegeneration. Common toxicities during treatment included fever, skin rash, arthralgia, and cytopenia. Combining therapy was well-tolerated and associated with clinical and molecular improvement in most patients with highly refractory systemic LCH and LCH-ND. Further prospective multi-center trials are required to optimize this approach, determine potential for combination therapy to achieve cure, and compare risks and benefits to chemotherapy or MAPK inhibitor monotherapy.

329MO HLX208, a novel BRAF V600E inhibitor, in adult patients with Langerhans cell histiocytosis and/or Erdheim-Chester disease harbouring BRAF V600E mutation

329MO HLX208, a novel BRAF V600E inhibitor, in adult patients with Langerhans cell histiocytosis and/or Erdheim-Chester disease harbouring BRAF V600E mutation

Quinazolin-4-one/3-cyanopyridin-2-one Hybrids as Dual Inhibitors of EGFR and BRAFV600E: Design, Synthesis, and Antiproliferative Activity.

A novel series of hybrid compounds comprising quinazolin-4-one and 3-cyanopyridin-2-one structures has been developed, with dual inhibitory actions on both EGFR and BRAFV600E. These hybrid compounds were tested in vitro against four different cancer cell lines. Compounds 8, 9, 18, and 19 inhibited cell proliferation significantly in the four cancer cells, with GI50 values ranging from 1.20 to 1.80 µM when compared to Doxorubicin (GI50 = 1.10 µM). Within this group of hybrids, compounds 18 and 19 exhibited substantial inhibition of EGFR and BRAFV600E. Molecular docking investigations provided confirmation that compounds 18 and 19 possess the capability to inhibit EGFR and BRAFV600E. Moreover, computational ADMET prediction indicated that most of the newly synthesized hybrids have low toxicity and minimal side effects.

Design and Synthesis of Novel 2-Acetamido, 6-Carboxamide Substituted Benzothiazoles as Potential BRAFV600E Inhibitors - In vitro Evaluation of their Antiproliferative Activity.

The oncogenic BRAFV600E kinase leads to abnormal activation of the MAPK signaling pathway and thus, uncontrolled cellular proliferation and cancer development. Based on our previous virtual screening studies which issued 2-acetamido-1,3 benzothiazole-6-carboxamide scaffold as active pharmacophore displaying selectivity against the mutated BRAF, eleven new substituted benzothiazole derivatives were designed and synthesized by coupling of 2-acetamidobenzo[d]thiazole-6-carboxylic acid with the appropriate amines in an effort to provide even more efficient inhibitors and tackle drug resistance often developed during cancer treatment. All derived compounds bore the benzothiazole scaffold substituted at position-2 by an acetamido moiety and at position-6 by a carboxamide functionality, the NH moiety of which was further linked through an alkylene linker to a sulfonamido (or amino) aryl (or alkyl) functionality or a phenylene linker to a sulfonamido aromatic (or non-aromatic) terminal pharmacophore in the order -C6 H4 -NHSO2 -R or reversely -C6 H4 -SO2 N(H)-R. These analogs were subsequently biologically evaluated as potential BRAFV600E inhibitors and antiproliferative agents in several colorectal cancer and melanoma cell lines. In all assays applied, one analog, namely 2-acetamido-N-[3-(pyridin-2-ylamino)propyl]benzo[d]thiazole-6-carboxamide (22), provided promising results in view of its use in drug development.

Combination BRAFV600E Inhibition with the Multitargeting Tyrosine Kinase Inhibitor Axitinib Shows Additive Anticancer Activity in BRAFV600E-Mutant Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is uniformly lethal. BRAFV600E mutation is present in 45% of patients with ATC. Targeted therapy with combined BRAF and MEK inhibition in BRAFV600E-mutant ATC can be effective, but acquired resistance is common because this combination targets the same pathway. Drug matrix screening, in BRAFV600E ATC cells, of highly active compounds in combination with BRAF inhibition showed multitargeting tyrosine kinase inhibitors (MTKIs) had the highest synergistic/additive activity. Thus, we hypothesized that the combination of BRAFV600E inhibition and an MTKI is more effective than a single drug or combined BRAF and MEK inhibition in BRAFV600E-mutant ATC. We evaluated the effect of BRAFV600E inhibitors in combination with the MTKI axitinib and its mechanism(s) of action. Methods: We evaluated the effects of BRAFV600E inhibitors and axitinib alone and in combination in in vitro and in vivo models of BRAFV600E-mutant and wild-type ATC. Results: The combination of axitinib and BRAFV600E inhibitors (dabrafenib and PLX4720) showed an additive effect on inhibiting cell proliferation based on the Chou-Talalay algorithm in BRAFV600E-mutant ATC cell lines. This combination also significantly inhibited cell invasion and migration (p < 0.001) compared with the control. Dabrafenib and PLX4720 arrested ATC cells in the G0/G1 phase. Axitinib arrested ATC cells in the G2/M phase by decreasing phosphorylation of aurora kinase B (Thr232) and histone H3 (Ser10) proteins and by upregulating the c-JUN signaling pathway. The combination of BRAF inhibition and axitinib significantly inhibited tumor growth and was associated with improved survival in an orthotopic ATC model. Conclusions: The novel combination of axitinib and BRAFV600E inhibition enhanced anticancer activity in in vitro and in vivo models of BRAFV600E-mutant ATC. This combination may have clinical utility in BRAFV600E-mutant ATC that is refractory to current standard therapy, namely combined BRAF and MEK inhibition.

A phase I, single-center, open-label study to investigate the absorption, distribution, metabolism and excretion of encorafenib following a single oral dose of 100 mg [14 C] encorafenib in healthy male subjects.

Encorafenib is a novel kinase inhibitor of BRAF V600E as well as wild-type BRAF and CRAF and has received approval, in combination with binimetinib, to treat BRAF V600E or V600K mutation-positive unresectable or metastatic melanoma or in combination with cetuximab to treat BRAF V600E mutation-positive colorectal cancer. The absorption, distribution, metabolism and excretion (ADME) of encorafenib was studied by administering [14 C] encorafenib (100 mg containing 90 μCi of radiolabeled material) to 4 healthy male subjects (NCT01436656). Following a single oral 100-mg dose of [14 C] encorafenib to healthy male subjects, the overall recovery of radioactivity in the excreta was ≥93.9% in all four subjects, indicating that good mass balance was achieved. An equal mean of 47.2% for the radioactivity dose was eliminated in the feces and urine. The percentage of the dose eliminated in the feces (5.0%) and urine (1.8%) as unchanged encorafenib was minor. Metabolism was found to be the major clearance pathway (~88% of the recovered radioactive dose) for encorafenib in humans and is predominantly mediated through N-dealkylation of the isopropyl carbamic acid methyl ester to form the primary phase 1 direct metabolite M42.5 (LHY746). Oral absorption was estimated from the radioactive dose recovered in the urine (47.2%) and the total radioactive dose recovered in the feces as metabolites (39%). Based on these values and the assumptions that encorafenib and its metabolites are stable in feces, the fraction of oral absorption was estimated to be at least ~86%.

Solvent impact on the photochemical properties of anticancer drug PLX4720; a computational study

The advancement in pharmaceutical research has led to a successful era but limitations because of the drug's photochemical behavior triggered the researchers to explore the promising revolution. In terms of molecular properties rationalizing, the photosensitive drug photochemical response is essential for knowing the associated potential phototoxicity. It is the need of the hour to check the influence of the molecular microenvironment on photochemical and the physicochemical properties of the PLX4720 B-RAFV600E inhibitor. The geometry optimization was done with functional wB97XD. TD-DFT calculation has been performed to study the PLX4720 absorption spectra in different solvents and hydrogen bonding. The polarity-dependent spectral properties are indicative of the microenvironments that influenced the structural properties of PLX4720. In polar solvents, a notable enhancement is observed in the oscillation strength of HOMO → LUMO, the main electronic transition affects the photochemical properties of PLX4720. The charge delocalization is enhanced by these effects on the heteroatoms that comprise electron lone pairs. The inter-molecular hydrogen bonding among PLX4720 and the microenvironment study is also carried out. As a result, a substantial effect on the spectral properties of PLX4720 is reported. The physiochemical properties of PLX4720 complex are investigated using DMSO through hydrogen bonding interaction, NBO and DOS analyses. These results have exposed considerable changes in the structural properties of the PLX4720 which is an indication of the impact of intermolecular hydrogen bonding on photochemical properties. The results also reveal a notable charge density change in the selected atoms, leading to greater electronic transitions. The final results provided here give an insight into understanding about PLX4720 photochemical behavior at the molecular level in biologically mimicked microenvironments.

From a mutation to a drug

Malignant melanoma is a dangerous skin cancer, accounting for the majority of skin cancer-related deaths. Many patients with this cancer have the V600E mutation in the BRAF gene. This mutation causes constitutive activation of the MAPK/ERK signaling pathway, significantly contributing to the process of carcinogenesis. We discuss the drug design process on the example of a specific BRAF V600E inhibitor, vemurafenib. We begin with the most commonly used drug design methods. The second part of the article focuses on vemurafenib. We analyze the invention of this BRAF V600E inhibitor and its analogue as well as the course of three stages of clinical trials. Then we provide information about other popular drugs for malignant melanoma, i.e. dacarbazine, ipilimumab and dabrafenib, and about the advantages of therapy with the simultaneous use of two inhibitors. Finally, we briefly discuss the role of artificial intelligence in the future of drug design.

Synthesis, Antioxidant and Antiproliferative Actions of 4-(1,2,3-Triazol-1-yl)quinolin-2(1H)-ones as Multi-Target Inhibitors.

The reaction of 4-azido-quinolin-2(1H)-ones 1a-e with the active methylene compounds pentane-2,4-dione (2a), 1,3-diphenylpropane-1,3-dione (2b), and K2CO3 was investigated in this study. This approach afforded 4-(1,2,3-triazol-1-yl)quinolin-2(1H)-ones 3a-j in high yields and purity. All newly synthesized products' structures were identified. Compounds 3a-j were tested for antiproliferative activity against a panel of four cancer cell lines. In comparison to the reference erlotinib (GI50 = 33), compounds 3f-j were the most potent derivatives, with GI50 values ranging from 22 nM to 31 nM. The most effective antiproliferative derivatives, 3f-j, were subsequently investigated as possible multi-target inhibitors of EGFR, BRAFV600E, and EGFRT790M. Compound 3h was the most potent inhibitor of the studied molecular targets, with IC50 values of 57 nM, 68 nM, and 9.70 nM, respectively. The apoptotic assay results demonstrated that compounds 3g and 3h function as caspase-3, 8, and Bax activators as well as down-regulators of the antiapoptotic Bcl2, and hence can be classified as apoptotic inducers. Finally, compounds 3g and 3h displayed promising antioxidant activity at 10 µM, with DPPH radical scavenging of 70.6% and 73.5%, respectively, compared to Trolox (77.6%).

Ezrin Inhibition Overcomes Acquired Resistance to Vemurafenib in BRAFV600E-Mutated Colon Cancer and Melanoma Cells In Vitro.

Despite the advancements in targeted therapy for BRAFV600E-mutated metastatic colorectal cancer (mCRC), the development of resistance to BRAFV600E inhibition limits the response rate and durability of the treatment. Better understanding of the resistance mechanisms to BRAF inhibitors will facilitate the design of novel pharmacological strategies for BRAF-mutated mCRC. The aim of this study was to identify novel protein candidates involved in acquired resistance to BRAFV600E inhibitor vemurafenib in BRAFV600E-mutated colon cancer cells using an integrated proteomics approach. Bioinformatic analysis of obtained proteomics data indicated actin-cytoskeleton linker protein ezrin as a highly ranked protein significantly associated with vemurafenib resistance whose overexpression in the resistant cells was additionally confirmed at the gene and protein level. Ezrin inhibition by NSC305787 increased anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant cells in an additive manner, which was accompanied by downregulation of CD44 expression and inhibition of AKT/c-Myc activities. We also detected an increased ezrin expression in vemurafenib-resistant melanoma cells harbouring the BRAFV600E mutation. Importantly, ezrin inhibition potentiated anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant melanoma cells in a synergistic manner. Altogether, our study suggests a role of ezrin in acquired resistance to vemurafenib in colon cancer and melanoma cells carrying the BRAFV600E mutation and supports further pre-clinical and clinical studies to explore the benefits of combined BRAF inhibitors and actin-targeting drugs as a potential therapeutic approach for BRAFV600E-mutated cancers.