Efficacy of BRAF/MEK-inhibitor therapy for epithelioid glioblastoma with a novel BRAFV600 mutation.

Epithelioid glioblastoma (eGB), a very aggressive and rare brain tumour, is associated with a dismal median overall survival. Effective therapies for patients with eGB, particularly with leptomeningeal dissemination, are still lacking. Here, we describe a case of a 25-year-old male diagnosed with an intramedullary cervical tumour with subsequent leptomeningeal disease. Histopathology identified a highly necrotising, epithelioid-type tumour with high cell density, most compatible with the diagnosis of an eGB. DNA analysis revealed an unprecedented B-Raf protooncogene, serine/threonine kinase (BRAF) gene variant in exon 15 (ENST00000288602.6, c.1799_1810delinsATG, p.(V600_W604delinsDG)), triggering activation of the mitogen-activated protein kinase (MAPK) pathway. Consequently, we initiated MAPK inhibitor (MAPKi) therapy, utilizing a combination of BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors. Liquid chromatography-tandem mass spectrometry analysis confirmed the drugs' presence in the patient's cerebrospinal fluid, indicating their capacity to cross the blood-brain barrier. Remarkably, the patient responded very well to therapy and transitioned from a near-comatose state to significantly improved health, sustained for over three months. This study highlights that MAPKi, particularly targeted towards novel BRAFV600 mutations, might offer promising advancements in eGB treatment strategies.

Characterization of two melanoma cell lines resistant to BRAF/MEK inhibitors (vemurafenib and cobimetinib).

BACKGROUND: BRAF (v-raf murine sarcoma viral oncogene homolog B1)/MEK (mitogen-activated protein kinase kinase) inhibitors are used for melanoma treatment. Unfortunately, patients treated with this combined therapy develop resistance to treatment quite quickly, but the mechanisms underlying this phenomenon are not yet fully understood. Here, we report and characterize two melanoma cell lines (WM9 and Hs294T) resistant to BRAF (vemurafenib) and MEK (cobimetinib) inhibitors. METHODS: Cell viability was assessed via the XTT test. The level of selected proteins as well as activation of signaling pathways were evaluated using Western blotting. The expression of the chosen genes was assessed by RT-PCR. The distribution of cell cycle phases was analyzed by flow cytometry, and confocal microscopy was used to take photos of spheroids. The composition of cytokines secreted by cells was determined using a human cytokine array. RESULTS: The resistant cells had increased survival and activation of ERK kinase in the presence of BRAF/MEK inhibitors. The IC50 values for these cells were over 1000 times higher than for controls. Resistant cells also exhibited elevated activation of AKT, p38, and JNK signaling pathways with increased expression of EGFR, ErbB2, MET, and PDGFRß receptors as well as reduced expression of ErbB3 receptor. Furthermore, these cells demonstrated increased expression of genes encoding proteins involved in drug transport and metabolism. Resistant cells also exhibited features of epithelial-mesenchymal transition and cancer stem cells as well as reduced proliferation rate and elevated cytokine secretion. CONCLUSIONS: In summary, this work describes BRAF/MEK-inhibitor-resistant melanoma cells, allowing for better understanding the underlying mechanisms of resistance. The results may thus contribute to the development of new, more effective therapeutic strategies.

Rapid but nondurable response of a BRAF exon 15 double-mutated spindle cell sarcoma to a combination of BRAF and MEK inhibitors.

INTRODUCTION: BRAF V600E substitution predicts sensitivity of a cancer to BRAF inhibitor therapy. The mutation is rarely found in soft-tissue sarcomas. Here we describe a case of undifferentiated spindle cell sarcoma showing primary insensitivity to standard chemotherapy and pronounced but non-sustained response to BRAF/MEK inhibitors at recurrence. CASE PRESENTATION: A 13-year-old girl was diagnosed with low-grade spindle cell sarcoma of pelvic localization, BRAF exon 15 double-mutated: c.1799T>A p.V600E and c.1819T>A p.S607T in cis-position. The tumor showed resistance to CWS-based first-line chemotherapy and was treated surgically by radical resection. Seven months after surgery the patient developed metastatic relapse with abdominal carcinomatosis. Combined targeted therapy with BRAF/MEK inhibitors afforded complete response in 1 month and was continued, though complicated by severe side effects (fever, rash) necessitating 1-2 week toxicity breaks. After 4 months from commencement the disease recurred and anti-BRAF/MEK regimen consolidation was unsuccessful. Intensive salvation chemotherapy was ineffective. Empirical immunotherapy afforded a transient partial response giving way to fatal progression with massive, abdominal cocoon-complicated peritoneal carcinomatosis. CONCLUSION: This is the first report of spindle cell sarcoma BRAF V600E/S607T double-mutated, responding to a combination of B-Raf and MEK inhibitors. Despite the low histological grade and radical surgical treatment of the tumor at primary manifestation, the disease had aggressive clinical course and the response to BRAF/MEK targeted therapy at recurrence was complete but nondurable. Empirical use of pembrolizumab provided no unambiguous evidence on the clinical relevance of immunotherapy in protein kinase -rearranged spindle cell tumors.

Toxicities from BRAF and MEK Inhibitors: Strategies to Maximize Therapeutic Success.

PURPOSE OF REVIEW: This report highlights several of the recent therapeutic advancements in the treatment of BRAF-mutant tumors, discusses the most common adverse events observed with BRAF-targeted agents, and suggests strategies to manage and mitigate treatment-related toxicities. RECENT FINDINGS: BRAF and MEK inhibitors represent a significant advancement in the treatment of BRAF-mutated malignancies with data across tumor types demonstrating the anti-tumor efficacy of dual MAPK inhibition. Although these agents have a reasonable toxicity profile, variable side effects across organ systems can develop. The discovery of activating BRAF mutations and subsequent development of BRAF and MEK inhibitors has transformed the treatment algorithms of BRAF-mutant malignancies. With increased application of these targeted regimens, identification and prompt management of their unique adverse events are crucial.

MEK-SHP2 inhibition prevents tibial pseudarthrosis caused by NF1 loss in Schwann cells and skeletal stem/progenitor cells.

Congenital pseudarthrosis of the tibia (CPT) is a severe pathology marked by spontaneous bone fractures that fail to heal, leading to fibrous nonunion. Half of patients with CPT are affected by the multisystemic genetic disorder neurofibromatosis type 1 (NF1) caused by mutations in the NF1 tumor suppressor gene, a negative regulator of RAS-mitogen-activated protein kinase (MAPK) signaling pathway. Here, we analyzed patients with CPT and Prss56-Nf1 knockout mice to elucidate the pathogenic mechanisms of CPT-related fibrous nonunion and explored a pharmacological approach to treat CPT. We identified NF1-deficient Schwann cells and skeletal stem/progenitor cells (SSPCs) in pathological periosteum as affected cell types driving fibrosis. Whereas NF1-deficient SSPCs adopted a fibrotic fate, NF1-deficient Schwann cells produced critical paracrine factors including transforming growth factor-ß and induced fibrotic differentiation of wild-type SSPCs. To counteract the elevated RAS-MAPK signaling in both NF1-deficient Schwann cells and SSPCs, we used MAPK kinase (MEK) and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibitors. Combined MEK-SHP2 inhibition in vivo prevented fibrous nonunion in the Prss56-Nf1 knockout mouse model, providing a promising therapeutic strategy for the treatment of fibrous nonunion in CPT.

Triple Blockade of Oncogenic RAS Signaling Using KRAS and MEK Inhibitors in Combination with Irradiation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of human malignancies and carries an exceptionally poor prognosis. It is mostly driven by multiple oncogenic alterations, with the highest mutation frequency being observed in the KRAS gene, which is a key oncogenic driver of tumorogenesis and malignant progression in PDAC. However, KRAS remained undruggable for decades until the emergence of G12C mutation specific KRAS inhibitors. Despite this development, this therapeutic approach to target KRAS directly is not routinely used for PDAC patients, with the reasons being the rare presence of G12C mutation in PDAC with only 1-2% of occurring cases, modest therapeutic efficacy, activation of compensatory pathways leading to cell resistance, and absence of effective KRASG12D or pan-KRAS inhibitors. Additionally, indirect approaches to targeting KRAS through upstream and downstream regulators or effectors were also found to be either ineffective or known to cause major toxicities. For this reason, new and more effective treatment strategies that combine different therapeutic modalities aiming at achieving synergism and minimizing intrinsic or adaptive resistance mechanisms are required. In the current work presented here, pancreatic cancer cell lines with oncogenic KRAS G12C, G12D, or wild-type KRAS were treated with specific KRAS or SOS1/2 inhibitors, and therapeutic synergisms with concomitant MEK inhibition and irradiation were systematically evaluated by means of cell viability, 2D-clonogenic, 3D-anchorage independent soft agar, and bioluminescent ATP assays. Underlying pathophysiological mechanisms were examined by using Western blot analyses, apoptosis assay, and RAS activation assay.

Targeting BRAF pathway in low-grade serous ovarian cancer.

Mutations in genes encoding for proteins along the RAS-RAF-MEK-ERK pathway have been detected in a variety of tumor entities including ovarian carcinomas. In the recent years, several inhibitors of this pathway have been developed, whose antitumor potential is currently being assessed in different clinical trials. Low grade serous ovarian carcinoma, is a rare gynecological tumor which shows favorable overall survival, compared to the general ovarian cancer population, but worrying resistance to conventional chemotherapies. The clinical behavior of low grade serous ovarian carcinoma reflects the different gene profile compared to high-grade serous carcinoma: KRAS/BRAF mutations. BRAF inhibitors as single agents were approved for the treatment of BRAF mutated tumors. Nevertheless, many patients face progressive disease. The understanding of the mechanisms of resistance to BRAF inhibitors therapy and preclinical studies showing that BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors combined therapy delays the onset of resistance compared to BRAF inhibitor single agent, led to the clinical investigation of combined therapy. The aim of this paper is to review the efficacy and safety of the combination of BRAF plus MEK inhibitors on ovarian carcinomas, in particularly focusing on low grade serous ovarian carcinoma.

Effect of NFX-179 MEK inhibitor on cutaneous neurofibromas in persons with neurofibromatosis type 1.

This phase 2a trial investigated the efficacy of NFX-179 Topical Gel, a metabolically labile MEK inhibitor, in the treatment of cutaneous neurofibromas (cNFs) in neurofibromatosis type 1. Forty-eight participants were randomized to four treatment arms: NFX-179 Topical Gel 0.05%, 0.15%, and 0.5% or vehicle applied once daily to five target cNFs for 28 days. Treatment with NFX-179 Topical Gel resulted in a dose-dependent reduction in p-ERK levels in cNFs at day 28, with a 47% decrease in the 0.5% NFX-179 group compared to the vehicle (P = 0.0001). No local or systemic toxicities were observed during the treatment period, and systemic concentrations of NFX-179 remained below 1 ng/ml. In addition, 20% of cNFs treated with 0.5% NFX-179 Topical Gel showed a ≥50% reduction in volume compared to 6% in the vehicle group by ruler measurement with calculated volume (P = 0.021). Thus, NFX-179 Topical Gel demonstrated significant inhibition of MEK in cNF with excellent safety and potential therapeutic benefit.

ALDH1A1 confers resistance to RAF/MEK inhibitors in melanoma cells by maintaining stemness phenotype and activating PI3K/AKT signaling.

The mitogen-activated protein kinase (MAPK/ERK) pathway is pivotal in controlling the proliferation and survival of melanoma cells. Several mutations, including those in BRAF, exhibit an oncogenic effect leading to increased cellular proliferation. As a result, the combination therapy of a MEK inhibitor with a BRAF inhibitor demonstrated higher efficacy and lower toxicity than BRAF inhibitor alone. This combination has become the preferred standard of care for tumors driven by BRAF mutations. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a known marker of stemness involved in drug resistance in several type of tumors, including melanoma. This study demonstrates that melanoma cells overexpressing ALDH1A1 displayed resistance to vemurafenib and trametinib through the activation of PI3K/AKT signaling instead of MAPK axis. Inhibition of PI3K/AKT signaling partially rescued sensitivity to the drugs. Consistently, pharmacological inhibition of ALDH1A1 activity downregulated the activation of AKT and partially recovered responsiveness to vemurafenib and trametinib. We propose ALDH1A1 as a new potential target for treating melanoma resistant to MAPK/ERK inhibitors.

RAF and MEK Inhibitors in Non-Small Cell Lung Cancer.

Lung cancer, despite recent advancements in survival rates, represents a significant global health burden. Non-small cell lung cancer (NSCLC), the most prevalent type, is driven largely by activating mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and receptor tyrosine kinases (RTKs), and less in v-RAF murine sarcoma viral oncogene homolog B (BRAF) and mitogen-activated protein-kinase kinase (MEK), all key components of the RTK-RAS-mitogen-activated protein kinase (MAPK) pathway. Learning from melanoma, the identification of BRAFV600E substitution in NSCLC provided the rationale for the investigation of RAF and MEK inhibition as a therapeutic strategy. The regulatory approval of two RAF-MEK inhibitor combinations, dabrafenib-trametinib, in 2017, and encorafenib-binimetinib, in 2023, signifies a breakthrough for the management of BRAFV600E-mutant NSCLC patients. However, the almost universal emergence of acquired resistance limits their clinical benefit. New RAF and MEK inhibitors, with distinct biochemical characteristics, are in preclinical and clinical development. In this review, we aim to provide valuable insights into the current state of RAF and MEK inhibition in the management of NSCLC, fostering a deeper understanding of the potential impact on patient outcomes.

Exceptional Response of BRAFV600E-Mutated Acinar Cell CUP to BRAF/MEK Inhibition.

Complete remission of BRAF V600E-driven ACC CUP by BRAF/MEK inhibition underscores importance of precision oncology.

The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers.

Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.

Ocular Effects of MEK Inhibitor Therapy: Literature Review, Clinical Presentation, and Best Practices for Mitigation.

MEK signaling pathway targeting has emerged as a valuable addition to the options available for the treatment of advanced cancers including melanoma and non-small cell lung cancer. Ophthalmologic monitoring of patients taking part in clinical trials of MEK inhibitors has shown that while ocular effects are common, generally emerging during the first days to weeks of treatment, the majority are either asymptomatic or have minimal visual impact and are benign, resolving without intervention or the need to reduce or stop MEK inhibitor therapy. However rare cases of serious, potentially vision-threatening ocular toxicities have been reported during MEK inhibitor therapy. There is currently no recommendation for routine ophthalmologic screening or monitoring of patients with advanced cancer who are initiating MEK inhibitor therapy. However, baseline ophthalmologic examination may be useful for all patients initiating MEK inhibitor therapy to allow the differentiation of preexisting pathology versus the development of MEK inhibitor-associated retinopathy in the event of the emergence of symptomatic ocular events. Regular ophthalmologic examination may be appropriate for patients at increased risk for ocular events, such as patients with a history of ocular inflammation, infection, or underlying macular/retinal disease. All patients reporting visual disturbance should be referred for prompt ophthalmologic review to determine the potential seriousness of any underlying abnormalities and whether there is a need for treatment modification or specific intervention. Understanding the potential consequences of ocular toxicities is of particular importance in the context of decision-making for the continuation of potentially life-prolonging medications such as MEK inhibitors.

Near complete remission of an inoperable pancreatic acinar cell carcinoma after BRAF-/MEK-inhibitor treatment-A case report and review of the literature.

INTRODUCTION: Pancreatic acinar cell carcinomas are rare malignant neoplasms. High-quality evidence about the best treatment strategy is lacking. We present the case of a 52-year-old male with a BRAFV600E -mutated PACC who experienced a complete remission after chemotherapy with BRAF-/MEK-inhibitors. CASE: The patient presented with upper abdomen pain, night sweat, and weight loss. CT scan showed a pancreatic tumor extending from the pancreas head to body. Histological workup identified an acinar cell carcinoma. As the tumor was inoperable, chemotherapy with FOFIRNIOX was initiated and initially showed a slight regression of disease. The regimen had to be discontinued due to severe side effects. Molecular analysis identified a BRAFV600E mutation, so the patient was started on BRAF- and MEK-inhibitors (dabrafenib/trametinib). After 16 months, CT scans showed a near complete remission with a markedly improved overall health. DISCUSSION: Studies suggest that up to one-fourth of PACCs carry a BRAF mutation and might therefore be susceptible to a BRAF-/MEK-inhibitor therapy. This offers a new therapeutic pathway to treat this rare but malignant neoplasm.

Dual p38MAPK and MEK inhibition disrupts adaptive chemoresistance in mesenchymal glioblastoma to temozolomide.

BACKGROUND: Precision treatment of glioblastoma is increasingly focused on molecular subtyping, with the mesenchymal subtype particularly resistant to temozolomide. Here, we aim to develop a targeted therapy for temozolomide resensitization in the mesenchymal subtype. METHODS: We integrated kinomic profiles and kinase inhibitor screens from patient-derived proneural and mesenchymal glioma-propagating cells and public clinical datasets to identify key protein kinases implicated in temozolomide resistance. RNAseq, apoptosis assays, and comet assays were used to examine the role of p38MAPK signaling and adaptive chemoresistance in mesenchymal cells. The efficacy of dual p38MAPK and MEK/ERK inhibition using ralimetinib (selective orally active p38MAPK inhibitor; phase I/II for glioblastoma) and binimetinib (approved MEK1/2 inhibitor for melanoma; phase II for high-grade glioma) in primary and recurrent mesenchymal tumors was evaluated using an intracranial patient-derived tumor xenograft model, focusing on survival analysis. RESULTS: Our transcriptomic-kinomic integrative analysis revealed p38MAPK as the prime target whose gene signature enables patient stratification based on their molecular subtypes and provides prognostic value. Repurposed p38MAPK inhibitors synergize favorably with temozolomide to promote intracellular retention of temozolomide and exacerbate DNA damage. Mesenchymal cells exhibit adaptive chemoresistance to p38MAPK inhibition through a pH-/calcium-mediated MEK/ERK pathway. Dual p38MAPK and MEK inhibition effectively maintain temozolomide sensitivity in primary and recurrent intracranial mesenchymal glioblastoma xenografts. CONCLUSIONS: Temozolomide resistance in mesenchymal glioblastoma is associated with p38MAPK activation. Adaptive chemoresistance in p38MAPK-resistant cells is mediated by MEK/ERK signaling. Adjuvant therapy with dual p38MAPK and MEK inhibition prolongs temozolomide sensitivity, which can be developed into a precision therapy for the mesenchymal subtype.

BRAF/MEK inhibitor rechallenge in advanced melanoma patients.

BACKGROUND: Effectivity of BRAF(/MEK) inhibitor rechallenge has been described in prior studies. However, structured data are largely lacking. METHODS: Data from all advanced melanoma patients treated with BRAFi(/MEKi) rechallenge were retrieved from the Dutch Melanoma Treatment Registry. The authors analyzed objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) for both first treatment and rechallenge. They performed a multivariable logistic regression and a multivariable Cox proportional hazards model to assess factors associated with response and survival. RESULTS: The authors included 468 patients in the largest cohort to date who underwent at least two treatment episodes of BRAFi(/MEKi). Following rechallenge, ORR was 43%, median PFS was 4.6 months (95% confidence interval [CI], 4.1-5.2), and median OS was 8.2 months (95% CI, 7.2-9.4). Median PFS after rechallenge for patients who discontinued first BRAFi(/MEKi) treatment due to progression was 3.1 months (95% CI, 2.7-4.0) versus 5.2 months (95% CI, 4.5-5.9) for patients who discontinued treatment for other reasons. Discontinuing first treatment due to progression and lactate dehydrogenase (LDH) levels greater than two times the upper limit of normal were associated with lower odds of response and worse PFS and OS. Symptomatic brain metastases were associated with worse survival, whereas a longer treatment interval between first treatment and rechallenge was associated with better survival. Responding to the first BRAFi(/MEKi) treatment was not associated with response or survival. CONCLUSIONS: This study confirms that patients benefit from rechallenge. Elevated LDH levels, symptomatic brain metastases, and discontinuing first BRAFi(/MEKi) treatment due to progression are associated with less benefit from rechallenge. A prolonged treatment interval is associated with more benefit from rechallenge.

BRAF-MEK Inhibition in Newly Diagnosed Papillary Craniopharyngiomas.

BACKGROUND: Craniopharyngiomas, primary brain tumors of the pituitary-hypothalamic axis, can cause clinically significant sequelae. Treatment with the use of surgery, radiation, or both is often associated with substantial morbidity related to vision loss, neuroendocrine dysfunction, and memory loss. Genotyping has shown that more than 90% of papillary craniopharyngiomas carry BRAF V600E mutations, but data are lacking with regard to the safety and efficacy of BRAF-MEK inhibition in patients with papillary craniopharyngiomas who have not undergone previous radiation therapy. METHODS: Eligible patients who had papillary craniopharyngiomas that tested positive for BRAF mutations, had not undergone radiation therapy previously, and had measurable disease received the BRAF-MEK inhibitor combination vemurafenib-cobimetinib in 28-day cycles. The primary end point of this single-group, phase 2 study was objective response at 4 months as determined with the use of centrally determined volumetric data. RESULTS: Of the 16 patients in the study, 15 (94%; 95% confidence interval [CI], 70 to 100) had a durable objective partial response or better to therapy. The median reduction in the volume of the tumor was 91% (range, 68 to 99). The median follow-up was 22 months (95% CI, 19 to 30) and the median number of treatment cycles was 8. Progression-free survival was 87% (95% CI, 57 to 98) at 12 months and 58% (95% CI, 10 to 89) at 24 months. Three patients had disease progression during follow-up after therapy had been discontinued; none have died. The sole patient who did not have a response stopped treatment after 8 days owing to toxic effects. Grade 3 adverse events that were at least possibly related to treatment occurred in 12 patients, including rash in 6 patients. In 2 patients, grade 4 adverse events (hyperglycemia in 1 patient and increased creatine kinase levels in 1 patient) were reported; 3 patients discontinued treatment owing to adverse events. CONCLUSIONS: In this small, single-group study involving patients with papillary craniopharyngiomas, 15 of 16 patients had a partial response or better to the BRAF-MEK inhibitor combination vemurafenib-cobimetinib. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT03224767.).

Vincristine Enhances the Efficacy of MEK Inhibitors in Preclinical Models of KRAS-mutant Colorectal Cancer.

Mutations in KRAS are found in more than 50% of tumors from patients with metastatic colorectal cancer (mCRC). However, direct targeting of most KRAS mutations is difficult; even the recently developed KRASG12C inhibitors failed to show significant benefit in patients with mCRC. Single agents targeting mitogen-activated protein kinase kinase (MEK), a downstream mediator of RAS, have also been ineffective in colorectal cancer. To identify drugs that can enhance the efficacy of MEK inhibitors, we performed unbiased high-throughput screening using colorectal cancer spheroids. We used trametinib as the anchor drug and examined combinations of trametinib with the NCI-approved Oncology Library version 5. The initial screen, and following focused validation screens, identified vincristine as being strongly synergistic with trametinib. In vitro, the combination strongly inhibited cell growth, reduced clonogenic survival, and enhanced apoptosis compared with monotherapies in multiple KRAS-mutant colorectal cancer cell lines. Furthermore, this combination significantly inhibited tumor growth, reduced cell proliferation, and increased apoptosis in multiple KRAS-mutant patient-derived xenograft mouse models. In vivo studies using drug doses that reflect clinically achievable doses demonstrated that the combination was well tolerated by mice. We further determined that the mechanism underlying the synergistic effect of the combination was due to enhanced intracellular accumulation of vincristine associated with MEK inhibition. The combination also significantly decreased p-mTOR levels in vitro, indicating that it inhibits both RAS-RAF-MEK and PI3K-AKT-mTOR survival pathways. Our data thus provide strong evidence that the combination of trametinib and vincristine represents a novel therapeutic option to be studied in clinical trials for patients with KRAS-mutant mCRC. SIGNIFICANCE: Our unbiased preclinical studies have identified vincristine as an effective combination partner for the MEK inhibitor trametinib and provide a novel therapeutic option to be studied in patients with KRAS-mutant colorectal cancer.

Kinetics of RTK activation determine ERK reactivation and resistance to dual BRAF/MEK inhibition in melanoma.

The combination of BRAF and MEK inhibitors (BRAFi/MEKi) has shown promising response rates in treating BRAF-mutant melanoma by inhibiting ERK activation. However, treatment efficacy is limited by the emergence of drug-tolerant persister cells (persisters). Here, we show that the magnitude and duration of receptor tyrosine kinase (RTK) activation determine ERK reactivation and persister development. Our single-cell analysis reveals that only a small subset of melanoma cells exhibits effective RTK and ERK activation and develops persisters, despite uniform external stimuli. The kinetics of RTK activation directly influence ERK signaling dynamics and persister development. These initially rare persisters form major resistant clones through effective RTK-mediated ERK activation. Consequently, limiting RTK signaling suppresses ERK activation and cell proliferation in drug-resistant cells. Our findings provide non-genetic mechanistic insights into the role of heterogeneity in RTK activation kinetics in ERK reactivation and BRAFi/MEKi resistance, suggesting potential strategies for overcoming drug resistance in BRAF-mutant melanoma.