BRAF Inhibitor Vemurafenib Research Articles

Ecto-NOX Disulfide-Thiol Exchanger 2 (ENOX2/tNOX) Is a Potential Prognostic Marker in Primary Malignant Melanoma and May Serve as a Therapeutic Target.

With an increasing incidence of malignant melanoma, new prognostic biomarkers for clinical decision making have become more important. In this study, we evaluated the role of ecto-NOX disulfide-thiol exchanger 2 (ENOX2/tNOX), a cancer- and growth-associated protein, in the prognosis and therapy of primary malignant melanoma. We conducted a tissue microarray analysis of immunohistochemical ENOX2 protein expression and The Cancer Genome Atlas (TCGA) ENOX2 RNA expression analysis, as well as viability assays and Western blots of melanoma cell lines treated with the ENOX2 inhibitor phenoxodiol (PXD) and BRAF inhibitor (BRAFi) vemurafenib. We discovered that high ENOX2 expression is associated with decreased overall (OS), disease-specific (DSS) and metastasis-free survival (MFS) in primary melanoma (PM) and a reduction in electronic tumor-infiltrating lymphocytes (eTILs). A gradual rise in ENOX2 expression was found with an increase in malignant potential from benign nevi (BNs) via PMs to melanoma metastases (MMs), as well as with an increasing tumor thickness and stage. These results highlight the important role of ENOX2 in cancer growth, progression and metastasis. The ENOX2 expression was not limited to malignant cell lines but could also be found in keratinocytes, fibroblasts and melanocytes. The viability of melanoma cell lines could be inhibited by PXD. A reduced induction of phospho-AKT under PXD could prevent the development of acquired BRAFi resistance. In conclusion, ENOX2 may serve as a potential prognostic marker and therapeutic target in malignant melanoma.

EGF/EGFR-YAP1/TEAD2 signaling upregulates STIM1 in vemurafenib resistant melanoma cells.

Stromal interaction molecule 1 (STIM1) is the endoplasmic reticulum Ca2+ sensor for store-operated calcium entry and is closely associated with carcinogenesis and tumor progression. Previously, we found that STIM1 is upregulated in melanoma cells resistant to the serine/threonine-protein kinase B-raf inhibitor vemurafenib, although the mechanism underlying this upregulation is unknown. Here, we show that vemurafenib resistance upregulates STIM1 through an epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR)-Yes-associated protein 1 (YAP1)/TEA domain transcription factor 2 (TEAD2) signaling axis. Vemurafenib resistance can lead to an increase in EGF and EGFR levels, causing activation of the EGFR signaling pathway, which promotes YAP1 nuclear localization to increase the expression of STIM1. Our findings not only reveal the mechanism by which vemurafenib resistance promotes STIM1 upregulation, but also provide a rationale for combined targeting of the EGF/EGFR-YAP1/TEAD2-STIM1 axis to improve the therapeutic efficacy of BRAF inhibitor in melanoma patients.

Multi-omics analysis delineates resistance mechanisms associated with BRAF inhibition in melanoma cells

Mutant BRAF is a critical oncogenic driver in melanoma, making it an attractive therapeutic target. However, the success of targeted therapy using BRAF inhibitors vemurafenib and dabrafenib has been limited due to development of resistance, restricting their clinical efficacy. A prior knowledge of resistance mechanisms to BRAFi or any cancer drug can lead to development of drugs that overcome resistance thus improving clinical outcomes. In vitro cellular models are powerful systems that can be utilized to mimic and study resistance mechanisms. In this study, we employed a multi-omics approach to characterize a panel of BRAF mutant melanoma cell lines to develop and systematically characterize BRAFi persister and resistant cells using exome sequencing, proteomics and phosphoproteomics. Our datasets revealed frequently observed intrinsic and acquired, genetic and non-genetic mechanisms of BRAFi resistance that have been studied in patients who developed resistance. In addition, we identified proteins that can be potentially targeted to overcome BRAFi resistance. Overall, we demonstrate that in vitro systems can be utilized not only to predict resistance mechanisms but also to identify putative therapeutic targets.

Peptostreptococcus stomatis promotes colonic tumorigenesis and receptor tyrosine kinase inhibitor resistance by activating ERBB2-MAPK

Peptostreptococcus stomatis (P.stomatis) is enriched in colorectal cancer (CRC), but its causality and translational implications in CRC are unknown. Here, we show that P.stomatis accelerates colonic tumorigenesis in ApcMin/+ and azoxymethane/dextran sodium sulfate (AOM-DSS) models by inducing cell proliferation, suppressing apoptosis, and impairing gut barrier function. P.stomatis adheres to CRC cells through its surface protein fructose-1,6-bisphosphate aldolase (FBA) that binds to the integrin α6/β4 receptor on CRC cells, leading to the activation of ERBB2 and the downstream MEK-ERK-p90 cascade. Blockade of the FBA-integrin α6/β4 abolishes ERBB2-mitogen-activated protein kinase (MAPK) activation and the protumorigenic effect of P.stomatis. P.stomatis-driven ERBB2 activation bypasses receptor tyrosine kinase (RTK) blockade by EGFR inhibitors (cetuximab, erlotinib), leading to drug resistance in xenograft and spontaneous CRC models of KRAS-wild-type CRC. P.stomatis also abrogates BRAF inhibitor (vemurafenib) efficacy in BRAFV600E-mutant CRC xenografts. Thus, we identify P.stomatis as an oncogenic bacterium and a contributory factor for non-responsiveness to RTK inhibitors in CRC.

Tumor eradication by triplet therapy with BRAF inhibitor, TLR 7 agonist, and PD-1 antibody for BRAF-mutated melanoma.

Programmed death 1 (PD-1)/programmed death-ligand 1 inhibitors are commonly used to treat various cancers, including melanoma. However, their efficacy as monotherapy is limited, and combination immunotherapies are being explored to improve outcomes. In this study, we investigated a combination immunotherapy involving an anti-PD-1 antibody that blocks the major adaptive immune-resistant mechanisms, a BRAF inhibitor that inhibits melanoma cell proliferation, and multiple primary immune-resistant mechanisms, such as cancer cell-derived immunosuppressive cytokines, and a Toll-like receptor 7 agonist that enhances innate immune responses that promote antitumor T-cell induction and functions. Using a xenogeneic nude mouse model implanted with human BRAF-mutated melanoma, a BRAF inhibitor vemurafenib was found to restore T-cell-stimulatory activity in conventional dendritic cells by reducing immunosuppressive cytokines, including interleukin 6, produced by human melanoma. Additionally, intravenous administration of the Toll-like receptor 7 agonist DSR6434 enhanced tumor growth inhibition by vemurafenib through stimulating the plasmacytoid dendritic cells/interferon-α/natural killer cell pathways and augmenting the T-cell-stimulatory activity of conventional dendritic cells. In a syngeneic mouse model implanted with murine BRAF-mutated melanoma, the vemurafenib and DSR6434 combination synergistically augmented the induction of melanoma antigen gp100-specific T cells and inhibited tumor growth. Notably, only triplet therapy with vemurafenib, DSR6434, and the anti-PD-1 antibody resulted in complete regression of SIY antigen-transduced BRAF-mutated melanoma in a CD8 T-cell-dependent manner. These findings indicate that a triple-combination strategy targeting adaptive and primary resistant mechanisms while enhancing innate immune responses that promote tumor-specific T cells may be crucial for effective tumor eradication.

A phase II study of tunlametinib in combination with vemurafenib in patients with BRAF V600-mutant advanced melanoma.

e21519 Background: The new MEK inhibitor tunlametinib combined with the BRAF inhibitor vemurafenib showed promising clinical activity in patients (pts) with BRAF V600-mutant solid tumors in a previous Phase I study (NCT03976050). Based on the result, we designed a phase IIa study to evaluate the efficacy and safety of tunlametinib plus vemurafenib in Chinese pts with BRAF V600 mutant melanoma. Methods: This open-label, single arm, multiple-center phase IIa study enrolled Chinese pts with BRAF V600E-mutant advanced melanoma, without restriction on the disease subtype (e.g., acral, mucosal, or cutaneous melanoma). Prior treatment was permitted, excluding BRAF/MEK inhibitors. Pts received oral tunlametinib at a dose of 9 mg twice daily and vemurafenib 720 mg twice daily. The primary endpoint was the objective response rate (ORR) per RECIST v1.1. Results: Between October 15, 2021, and June 22nd, 2022, 17 pts were enrolled and included in the analysis. Thirteen (76.5%) pts had received previous systemic antitumor therapy, with 12 (70.6%) receiving immunotherapy. Seven (41.2%) pts had acral melanoma, 8 (47.1%) pts had skin melanoma (non-acral) and 2 (11.8%) pts had mucosal melanoma. All pts had metastatic disease. With a median follow-up of 15.3 months (95%CI: 6.8; NE), 12 (70.6%) pts achieved confirmed partial response (PR), resulting in a confirmed ORR of 70.6% (95% CI: 44.0%-89.7%). The disease control rate was 100% (95% CI: 80.5%, 100.0%). The median progression-free survival (PFS) was 10.0 months (95% CI: 5. 5, NE). the duration of response (DOR) was 20.1 (95% CI: 4.2, NE). The overall survival (OS) was 21.6 months (95% CI: 14.5, NE), with a 12-months OS rate of 87.4% (95% CI: 58.1%, 96.7%). Subgroup analysis revealed that in pre-treated pts, the confirmed ORR was 76.9% (95% CI: 46.2%, 95.0%), the median PFS was 10.0 months (95% CI: 5.5, NE), while in previous untreated pts, the mPFS was 14.0 months (95% CI: 9.9, NE). In pts with skin melanoma, the confirmed ORR was 87.5% (47.3%, 99.7%), the mPFS was 10.0 months (95% CI: 5.5, NE), the mOS was 21.6 months (95% CI: 5.7, NE). In pts with acral melanoma, the ORR was 57.1% (95% CI: 18.4%,90.1%), the mPFS was 18.1 months (95% CI: 2.8, NE), the mOS not reached (12.3, NE). The most common adverse events included rash, pyrexia, and elevated blood creatin phosphokinase, most of which were of grade 1-2 severity. Grade 3 or higher TRAEs occurred in 12 (70.6%) pts, of which 41.2% were rash. Most AEs were manageable with supportive therapy or dose interruption. No treatment-related deaths were reported. Conclusions: The addition of a MEK inhibitor tunlametinib to vemurafenibexhibited promising clinical efficacy in Chinese patients with BRAF V600-mutant advanced melanoma, with a manageable safety profile. Additionally, notable effects were observed in pts with acral melanoma. The efficacy in pre-treated patients was comparable to that of similar agents in previous untreated patients. Clinical trial information: NCT05263453 .

Abstract 3020: The tumor suppressive role of methylthioadenosine phosphorylase in melanoma

Abstract Methylthioadenosine phosphorylase (MTAP) is an enzyme that is expressed in virtually all tissues but lost in many cancers. MTAP deficiency can be due to either deletion of the MTAP gene or methylation of the MTAP promoter. In normal cells, MTAP catalyzes the conversion of methylthioadenosine (MTA), produced during polyamine biosynthesis, to adenine and 5-methylthioribose-1-phosphate, which is subsequently converted to methionine. MTAP deficiency results in intracellular accumulation of MTA. Although recent genome wide association studies (GWAS) have associated the MTAP locus with melanoma risk, the molecular mechanisms linking MTAP loss to increased tumorigenesis are not yet fully understood. In this study, we hypothesized that loss of MTAP and the resulting accumulation of MTA would dysregulate microphthalmia transcription factor (MITF), a master transcriptional regulator of melanocytes that is also an amplified or mutated oncogene in melanoma. We identify a novel signaling mechanism in which MTA accumulation from MTAP deficiency aberrantly induces MITF expression via inhibition of the cAMP phosphodiesterase PDE4D3 in melanocytes. Inhibition of PKA abolishes this MITF induction, suggesting that the PKA pathway is hyperactivated upon MTAP deficiency. Downregulation of MTAP leads to increased proliferation of melanoma cells in vitro and increased melanoma tumor growth in vivo. We also show that MTAP deficient melanoma cells are more resistant to the BRAF inhibitor vemurafenib and observe MTAP downregulation in BRAF inhibitor resistant melanoma clinical specimens. Furthermore, MTAP downregulation via decreased expression and/or genomic loss corresponds to significantly worse outcomes in early-stage cutaneous melanomas from multiple datasets, thereby identifying a clinically important sub-category of “thin-lethal” tumors. Citation Format: Jennifer J. Hsiao, Pedro Andreu-Perez, Lajos V. Kemény, Miroslav Hejna, Jérémie Nsengimana, Jennifer Allouche, Abdullah Al Emran, D. Timothy Bishop, Göran B. Jönsson, Julia Newton-Bishop, Jun Song, David E. Fisher. The tumor suppressive role of methylthioadenosine phosphorylase in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3020.

A Rare Case of Hairy Cell Leukemia with COVID Infection: A Case Report

Abstract Hairy cell leukemia (HCL) is a distinct subset of chronic lymphoid leukemia, presents with unique features, including the characteristic "hairy" projections seen in neoplastic B cells. We reported a case on hairy cell leukemia (HCL), emphasizing the challenges posed by this rare hematological malignancy, especially in the context of concurrent health crises such as the COVID-19 pandemic. This malignancy, though uncommon, necessitates comprehensive diagnostic methodologies, such as peripheral blood studies, flow cytometry, and bone marrow biopsy. Treatment strategies for HCL involve tailored approaches based on symptomatic presentations. Purine analogs, cladribine and pentostatin, stand as first-line interventions, showcasing efficacy in inducing and sustaining remission. However, the prolonged immunosuppression resulting from these treatments warrants vigilant monitoring for potential infectious complications. Emerging therapies, such as the BRAF inhibitor vemurafenib, provide additional options for refractory or progressive cases. The post-treatment phase demands meticulous follow-up, with an emphasis on regular blood work assessments by an oncology nurse and primary care physician. Our case further highlights the need for heightened awareness among healthcare professionals, particularly in the post-COVID-19 phase, where persistent symptoms led to the discovery of an underlying hematological disorder. Keywords: Hairy cell leukemia, Evan syndrome, Covid-19.

Integrated Proteogenomics Uncover Mechanisms of Glioblastoma Evolution, Pointing to Novel Therapeutic Targets.

Nearly all glioblastoma (GBM) patients relapse following standard treatment and eventually succumb to disease. While large-scale, integrated multiomic studies have tremendously advanced the understanding of primary GBM at the cellular and molecular level, the posttherapeutic trajectory and biological properties of recurrent GBM remain poorly understood. This knowledge gap was addressed in a recent Cancer Cell article in which Kim and colleagues report on a highly integrative proteogenomic analysis performed on 123 matched primary and recurrent GBMs that uncovered a dramatic evolutionary shift from a proliferative state at initial diagnosis to the activation of neuronal and synaptogenic pathways at recurrence following therapy. Neuronal transition was characterized by posttranslational activation of WNT/PCP signaling and BRAF kinase, while many canonical oncogenic pathways, and EGFR in particular, were downregulated. Parallel multiomics analyses of patient-derived xenograft (PDX) models corroborated this evolutionary trajectory, allowing in vivo experiments for translational significance. Notably, targeting BRAF kinase disrupted both the neuronal transition and migration capabilities of recurrent gliomas, which were key characteristics of posttreatment progression. Furthermore, combining BRAF inhibitor vemurafenib with temozolomide prolonged survival in PDX models. Overall, the results reveal novel biological mechanisms of GBM evolution and therapy resistance, and suggest promising therapeutic intervention.

The use of BRAF-inhibitors as monotherapy and in combination with cytosine arabinoside and 2-chloro-2’deoxyadenosine in pediatric patients with different forms of Langerhans cell histiocytosis

Background. Langerhans cell histiocytosis (LCH) is a rare disease that occurs due to abnormal proliferation and expansion of myeloid precursors. The occurrence of mutations in genes that encode key kinases of MAPK-signaling pathway leads to its pathological activation and has been shown the cause of disease. Mutations in BRAF and MAP2K1 genes are the most frequent among LCH patients. The effectiveness of BRAF-inhibitors in LCH patients has been shown in numerous studies.The purpose of the study – analyze the experience of BRAF-inhibitor vemurafenib administration as monotherapy and in combination with cytosine arabinoside (ARA-C) and 2-chloro-2'-deoxyadenosine (2-CdA) in pediatric patients with different forms of LCH.Materials and methods. Fifteen patients with various forms of LCH were enrolled in the study. BRAF mutations were detected in 14 patients, mutation in the MAP2K1 gene was detected in one case. Patients with “risk organ” (RO) involvement were included in the first group (n = 9). These patients received combined therapy with vemurafenib and ARA-C/2-CdA. Patients without RO involvement, included in group 2 (n = 6), received vemurafenib as monotherapy. The assessment of the response to the therapy in group 1 was carried out in accordance with the DAS scale, in group 2 in accordance with the RECIST v1.1. The toxicity assessment in both groups was carried out in accordance with the CTCAE v5.0.Results. All patients in group 1 achieved non-active disease status with a median of 35 (28–61) days. In group 2 partial response to vemurafenib was achieved in 5 cases. Relapse after targeted therapy termination was diagnosed in two patients. Photodermatitis was the most common side effect of targeted therapy.Conclusions. The use of vemurafenib was effective in both groups. There were no cases of grade III–IV toxicity according to CTCAE v5.0 associated with vemurafenib administration in this study. The combination of vemurafenib and ARA-C/2-CdA showed high efficacy and good tolerability in group 1. Two cases of disease relapse after targeted therapy cessation in group 2 show that the monotherapy approach does not always allow to achieve long-term remission in LCH patients.

Vemurafenib induces senescence in acute myeloid leukemia and myelodysplastic syndrome by activating the HIPPO signaling pathway: implications for potential targeted therapy

BackgroundThe outcome of Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remain dismal despite the development of treatment. Targeted therapy is gaining more and more attention in improving prognosis.MethodsExpression of BRAF was analyzed by RT-qPCR in AML and MDS patients. Cells viability treated by drugs was measured by CCK-8 assay. Network pharmacology and RNA-sequence were used to analyze the mechanism of drugs and verified in vitro and xenograft tumor model.ResultsHere we showed that BRAF was overexpressed in AML and MDS patients, and correlated with poor prognosis. The BRAF inhibitor-Vemurafenib (VEM) could significantly induce senescence, proliferation inhibition and apoptosis in AML cells, which can be enhanced by Bortezomib (BOR). This inhibitory effect was also verified in CD34 + cells derived from AML patients. Mechanistically, we showed that VEM combined with BOR could turn on HIPPO signaling pathway, thereby inducing cellular senescence in AML cells and xenograft mouse.ConclusionsTaken together, our findings demonstrate a significant upregulation of BRAF expression in AML and MDS patients, which is associated with unfavorable clinical outcomes. We also discovered that the BRAF inhibitor Vemurafenib induces cellular senescence through activation of the HIPPO signaling pathway. Analysis of BRAF expression holds promise as a prognostic indicator and potential therapeutic target for individuals with AML and MDS.

BRAF and MEK inhibitor combinations induce potent molecular and immunological effects in NRAS-mutant melanoma cells: Insights into mode of action and resistance mechanisms.

About 25% of melanoma harbor activating NRAS mutations, which are associated with aggressive disease therefore requiring a rapid antitumor intervention. However, no efficient targeted therapy options are currently available for patients with NRAS-mutant melanoma. MEK inhibitors (MEKi) appear to display a moderate antitumor activity and also immunological effects in NRAS-mutant melanoma, providing an ideal backbone for combination treatments. In our study, the MEKi binimetinib, cobimetinib and trametinib combined with the BRAF inhibitors (BRAFi) encorafenib, vemurafenib and dabrafenib were investigated for their ability to inhibit proliferation, induce apoptosis and alter the expression of immune modulatory molecules in sensitive NRAS-mutant melanoma cells using two- and three-dimensional cell culture models as well as RNA sequencing analyses. Furthermore, NRAS-mutant melanoma cells resistant to the three BRAFi/MEKi combinations were established to characterize the mechanisms contributing to their resistance. All BRAFi induced a stress response in the sensitive NRAS-mutant melanoma cells thereby significantly enhancing the antiproliferative and proapoptotic activity of the MEKi analyzed. Furthermore, BRAFi/MEKi combinations upregulated immune relevant molecules, such as ICOS-L, components of antigen-presenting machinery and the "don't eat me signal" molecule CD47 in the melanoma cells. The BRAFi/MEKi-resistant, NRAS-mutant melanoma cells counteracted the molecular and immunological effects of BRAFi/MEKi by upregulating downstream mitogen-activated protein kinase pathway molecules, inhibiting apoptosis and promoting immune escape mechanisms. Together, our study reveals potent molecular and immunological effects of BRAFi/MEKi in sensitive NRAS-mutant melanoma cells that may be exploited in new combinational treatment strategies for patients with NRAS-mutant melanoma.

Abstract B164: ASTX029 is a dual mechanism ERK1/2 inhibitor with activity in models of MAPK-inhibitor resistance

Abstract Clinical efficacy of RAF, MEK1/2 (MEK) and KRASG12C inhibitors has been demonstrated in MAPK-activated cancers. However, response is often short-lived due to resistance mechanisms, which commonly confer reactivation of ERK1/2 (ERK). As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for overcoming resistance to upstream inhibition. ASTX029 is a potent and selective dual-mechanism ERK inhibitor. Due to its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK by MEK. It is currently in Phase 1/2 trial in patients with advanced solid tumors (NCT03520075). Here we demonstrate the potent activity of ASTX029 in models of acquired MAPK inhibitor resistance. Two MAPK inhibitor-resistant models were generated by continuous culture of A375 (BRAFV600E -mutant melanoma) cells in the presence of the BRAF inhibitor vemurafenib (A375R) or by introduction of an NRASQ61K mutation (A375-NRASQ61K). The A375-NRASQ61K and A375R cell lines were resistant to vemurafenib in cell proliferation assays (IC50 >3 µM and >10 µM respectively, compared to IC50 110 nM in parental A375 cells). They were also less sensitive to the MEK inhibitor selumetinib (IC50 270 nM and 1000 nM, respectively, compared to IC50 43 nM in parental A375). Both models were highly sensitive to ASTX029, with proliferation assay IC50 values of 12 nM (A375-NRASQ61K) and 7.2 nM (A375R), values which were not significantly different to the ASTX029 IC50 of parental A375. ASTX029 inhibited MAPK signalling in both models, whereas vemurafenib and selumetinib had no effect. Treatment of A375R tumour-bearing mice with 50 mg/kg bid vemurafenib (which causes significant tumor growth inhibition in parental A375 xenografts), did not result in significant tumor growth inhibition. In contrast, 75 mg/kg qd ASTX029 conferred significant anti-tumor activity (P< 0.001). The acquisition of MEK mutations is a known resistance mechanism to RAF, MEK and KRASG12C inhibitors. We performed a CRISPR-tiling screen on MEK1 and identified mutations that conferred resistance to MEK inhibitors. The identified MEK1 mutations either caused MEK activation (indicated by elevated pERK levels) or were predicted to prevent MEK inhibitor binding (supported by MEK1 structural analysis). All mutations conferred resistance to selumetinib (7- to 200-fold increase in cell proliferation IC50 values relative to parental cells). Mutations predicted to prevent MEK inhibitor binding were sensitive to vemurafenib and the pan-RAF inhibitor LY3009120, whereas those that caused MEK1 activation were resistant (5- to 10-fold increase in cell proliferation IC50 relative to parental cells). However, they were all sensitive to ASTX029 (IC50 values of 4 to 17 nM). We conclude that the MAPK inhibitor resistance mechanisms described did not confer resistance to ASTX029. These data highlight the therapeutic potential of ASTX029 for the treatment of cancers, which have acquired resistance to inhibitors of upstream components of the MAPK pathway. Citation Format: Zhiqiang Zhang, Christopher Hindley, Andrea Biondo, Nicola Wallis, John Lyons, Joanne Munck. ASTX029 is a dual mechanism ERK1/2 inhibitor with activity in models of MAPK-inhibitor resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B164.

Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor.The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed.Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity.Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

Real-Life Efficacy and Safety of Vemurafenib Plus Rituximab (V+R) in Relapsed or Refractory Hairy Cell Leukemia: A Multi-Center Retrospective Study (HCL-PG03R)

BACKGROUND Hairy cell leukemia (HCL) is a BRAFV600E-mutated indolent B-cell neoplasm (Tiacci et al., NEJM 2011) often relapsing after chemotherapy with purine analogs. BRAF inhibition is very active in relapsed/refractory (R/R) HCL, but produces mostly partial remissions (PR) and never clears minimal residual disease (MRD) (Tiacci, Park, et al., NEJM 2015). In a recent phase-2 single-center study on 30 R/R patients (pts) with a median of 3 prior therapies, a short chemotherapy-free regimen of the BRAF inhibitor vemurafenib (V) + rituximab (R; Mabthera) led to 87% complete remissions (CR), 60% MRD-negativity and 85% relapse-free survival (RFS) at a median follow-up of 34 months, with a favorable toxicity profile (Tiacci et al., NEJM 2021). METHODS A multi-center retrospective study was conducted to validate in a real-world setting the off-label use of V+R in R/R HCL needing therapy due to cytopenia(s). RESULTS From 6/2019 to 12/2021, 35 pts (median age 56 years, range 42-80) were treated at 14 Italian centers with VR (V: 960 mg bid for 8 weeks; R: 375 mg/m2 every 2 weeks for 8 doses); biosimilar R was used in 89% of pts. Prior therapies (median 2; range 0-11) included chemotherapy with cladribine or pentostatin (94% of pts), interferon-alpha (29%), rituximab (31%), splenectomy (6%), BRAF inhibitor monotherapy (6%) and zanubrutinib (3%). Median blood counts across the 35 pts were: platelets 69000/mmc, neutrophils 860/mmc and hemoglobin 11.6 g/dl (with 20% of pts, i.e. 7/35, requiring transfusions). Eleven pts (31%) had active (n=3) or latent (n=8) infections, including: ongoing systemic atypical mycobacteriosis (n=1); ongoing miliary tuberculosis (n=1; a 80-year old pt who was the only one receiving V+R as first-line therapy); ongoing large cerebral abscess (n=1); HIV infection controlled by anti-viral therapy (n=1); and latent tuberculosis (n=1) or HBV (n=6) infections requiring anti-microbial prophylaxis. Toxicity was as expected, mostly of grade 1-2 and always reversible (Table), allowing high relative dose intensities (RDI) for both drugs (V, median 97%; interquartile range/IQR 73-100%; R, 100% in 85% of pts). Similarly, among the 33/35 (94%) pts who received V for a substantial duration (≥35 days), and were thus evaluable for dose density, 20 pts (61%) completed V without ever suspending the drug, including 14/33 pts (42%) without any dose reduction. Vemurafenib toxicities were mainly represented by cutaneous rash, asymptomatic liver and pancreatic laboratory abnormalities, arthralgia, warts and photosensitivity. Three pts (9%) were not evaluable for response as they died prematurely due to atypical mycobacteriosis, complications of neurosurgery for cerebral abscess and sudden death unrelated to study drugs. Recovery of platelets (≥100000/mmc), neutrophils (≥1500/mmc) and hemoglobin (≥ 11 g/dl) occurred quickly, after a median of 2, 4 and 7 weeks after starting treatment, respectively. A CR was observed in 30/32 evaluable cases (94%; or 86%, 30/35 pts, by intention to treat/ITT). CR was obtained in all evaluable cases refractory to chemotherapy (n=8) or rituximab (n=3), or previously splenectomized (n=2) or unable to reach CR with a prior BRAF inhibitor (n=2). The only 2 evaluable pts not achieving a CR (1 PR; 1 short-lived resolution of cytopenias) received V at only 16%/50% RDI and R at only 13%/34% RDI due to drug toxicities. Analysis of MRD by allele-specific PCR for BRAF-V600E in the bone marrow aspirate was performed in 30 pts and showed MRD clearance (<0.05% mutant alleles) at a high rate: 79% (22/28 CR pts; or 65%, 22/35 pts, by ITT), which raised to 82% (66% by ITT) when including 1 additional CR case untested by PCR which had negative bone marrow flow cytometry. At a median follow-up of 21 months after the end of treatment, RFS was high among the 31 responding pts, with just 1 relapse (3%) in the only case obtaining a PR post-therapy (Figure). CONCLUSIONS This study validates in a real-life context the high efficacy and tolerability of the V+R chemo-free regimen delivered to R/R HCL pts, including the cost-saving use of biosimilar rituximab.

BIOM-04. IMPROVEMENT OF LEPTOMENINGEAL DISEASE (LMD) WITH MEK AND BRAF INHIBITORS, CRANIOSPINAL IRRADIATION (CSI), AND INTRATHECAL (IT) CHEMOTHERAPY IN ANAPLASTIC PLEOMORPHIC XANTHOASTROCYTOMA (PXA)

Abstract Anaplastic pleomorphic xanthoastrocytomas (aPXA) are rare gliomas with a five-year survival of 57%. Standard treatment involves maximal safe resection followed by radiotherapy. BRAF mutation is frequently seen in these patients and is a promising target with concurrent BRAF and MEK inhibitors. However, there is no standard treatment for patients with aPXA and leptomeningeal disease (LMD). Literature review identified four cases with overall survivals (OS) of 3, 4, 18, and 66 months, respectively. We report a 23-year-old female who presented with headache, nausea, vomiting, and aphasia with MRI evidence of a left temporal lobe enhancing mass. Her tumor was initially diagnosed as epithelioid glioblastoma, WHO grade IV after craniotomy. She received concurrent radiation and temozolomide with subsequent 12 cycles of TMZ. After identification of BRAF V600E mutation by next generation sequencing, we revisited her diagnosis, and it was changed to aPXA. Three years later, she presented to the ER with aphasia, headache, weakness, nausea, and vomiting with MRI evidence of hydrocephalus and LMD by brain and spine MRI as well as cerebrospinal fluid analysis. Her treatment regimen consisted of the MEK inhibitor cobimetinib, the BRAF inhibitor vemurafenib, partial craniospinal irradiation (CSI), and intrathecal (IT) chemotherapy with methotrexate, topotecan, cytarabine, and thiotepa sequentially. She is currently doing well with mild bilateral leg weakness. She continues on therapy with BRAF and MEK inhibitors. Her OS is 56 months. Adverse events (AE) were tabulated according to the Common Terminology Criteria for Adverse Events Version 5.0 and included grade IV neutropenia, grade III anemia, grade II rash acneiform, and grade II nausea/vomiting. All AEs were manageable and reversible. Despite the limitations of a case report and retrospective review, our findings suggest that MEK and BRAF inhibitors, CSI, and IT chemotherapy may be effective treatment options for patients with aPXA and LMD.

Efficacy of Chemo-Free Combination Therapy with BRAF Inhibitor and Rituximab in Classic Hairy Cell Leukemia: Insights from the French Experience

Background: Hairy cell leukemia (HCL) is a rare form of leukemia. The introduction of purine analogs has revolutionized its prognosis, leading to a life expectancy closer to that of the general population. However, patients experience relapses and require other treatment options. The BRAF V600E mutation is present in 80-100% of patients with HCL, which has prompted the use of BRAF inhibitors (vemurafenib or dabrafenib) in this condition. Recently, the combination of vemurafenib with rituximab has shown efficacy with durable responses in relapsed patients. Here, we present, for the first time, real-life data on the combination of a BRAF inhibitor with rituximab through a retrospective study involving 9 French centers. Data collection was completed by July 1, 2023. Results: We collected data from 17 patients with a male-to-female ratio of 13:4; the median age was 69 years (38-90), and 6 patients were aged over 75. The median number of previous treatments was 4 (0-7), and 3 patients were treatment-naive because they were deemed ineligible for purine analogs based on age or performance status. All 14 relapsed patients had previously received purine analogs once or multiple times (1-3). The median progression-free survival (PFS) after purine analogs was 48 months, and the median PFS after the last line of therapy was 24 months. Five patients were exposed to moxetumumab pasudotox, and 3 patients received single-agent vemurafenib previously. The BRAF V600E mutation was tested and positive for 16 patients. In our cohort, 13 patients received a combination of vemurafenib with rituximab (V+R), and 4 patients received a combination of dabrafenib with rituximab (D+R). All patients received the same regimen (Tiacci, NEMJ 2021), which included an induction phase (V+R or D+R for 8 weeks) followed by rituximab consolidation (4 injections every 15 days), except for one patient who received a rituximab sequence followed by vemurafenib. At the time of analysis, 15 out of 17 patients had completed the treatment, and the remaining 2 had completed the induction phase. Fourteen patients initiated treatment at the standard dose (960 mg bid for vemurafenib and 150 mg bid for dabrafenib), and 3 patients at a reduced dose (240-480 mg bid for vemurafenib). Toxicity of any grade was reported in 15 out of 17 patients, with 3 of them experiencing grade 3-4 toxicity. Skin toxicity was the most frequent, recorded in 12 patients (grade 1-2), leading to temporary treatment interruption in 2 patients and dose reduction in 6 patients. Other toxicities included musculoskeletal, hepatic, hematological, and cardiac, with only one patient experiencing a grade 2 infection during treatment. No secondary cancers were reported during or after treatment. Treatment was discontinued due to toxicity in 5 patients, with 4 of them resuming treatment later. Eight patients required a dosage reduction, with a median final vemurafenib dosage of 480 mg bid. At the end of treatment, 14 patients achieved a complete hematological response, and 1 patient achieved a partial response. Among the 9 patients with early response data, the median time for platelet recovery was 15 days, and for neutrophil recovery, it was 14 days. End-of-treatment bone marrow aspiration and/or biopsy was performed for 8 patients, showing a complete response in 7 patients. With a median follow-up of 12 months (4-56), only 2 patients had progressed, and as of the last update, no patients had died, and the 2 relapsed patients achieved a complete response after a new line of therapy. Conclusion: Despite a limited median follow-up of 12 months, the combination of a BRAF inhibitor with rituximab proves to be an effective regimen in heavily treated patients with HCL, achieving a 100% overall response rate. Our study confirms a rapid hematological response and an acceptable toxicity profile, even in elderly patients. The most frequent toxicity observed was cutaneous toxicity, mainly grade 1-2, and it never led to a definitive treatment discontinuation. A comparison of the two BRAF inhibitors in terms of efficacy or toxicity cannot be established due to the small sample size of patients treated with dabrafenib. Lastly, the combination of rituximab with vemurafenib or dabrafenib appears to be an interesting option in frontline treatment for patients ineligible for purine analogs.

Efficacy and Safety of Obinutuzumab in Relapsed or Refractory Hairy Cell Leukemia (R/R HCL): An Italian Multicenter Phase-2 Academic Trial (HCL-PG04)

INTRODUCTION About 50% of HCL patients (pts) relapse after chemotherapy with purine analogs (PA). After identifying the BRAF-V600E mutation as the genetic cause of HCL (Tiacci et al. NEJM 2011), we documented a high efficacy of the BRAF inhibitor vemurafenib in R/R pts (Tiacci, Park et al. NEJM 2015), especially when added to rituximab (Tiacci et al. NEJM 2021). HCL expresses bright CD20 and obinutuzumab (OBI) is a more effective anti-CD20 agent than rituximab (RTX) in other indolent B-cell neoplasms such as chronic lymphocytic leukemia and follicular lymphoma. RTX produces ~20% complete remissions (CR) in R/R HCL (Kreitman, ASH Educ Program 2012), while OBI activity in this setting is unknown. METHODS In the ongoing academic, phase-2, multi-center trial HCL-PG04, R/R HCL pts needing treatment due to cytopenia(s) received OBI 1000 mg intravenously on days 1-8-15 of cycle 1 and on day 1 of cycles 2-6 (1 cycle = 28 days). CR required resolution of cytopenias (platelets ≥ 100,000/mmc; neutrophils ≥ 1500/mmc; hemoglobin ≥ 11 g/dl), no palpable splenomegaly and no leukemic cells on morphologic analysis of the bone marrow biopsy, as assessed one month after the last OBI dose. Minimal residual disease (MRD) was analyzed by BRAF-V600E specific PCR in the bone marrow aspirate (sensitivity: 0.05% mutant alleles). RESULTS We enrolled 26 pts (M/F 24/2; median age 62 years, range 39-80) with a median of 2 prior therapies (range 1-7), including 7 refractory to PA (26%) and 2 to RTX (8%). Median values of disease burden parameters at baseline were: 70% BM HCL infiltration, 830 neutrophils/mmc, 62000 platelets/mmc, 14 g/dl hemoglobin, and 16 cm of longest spleen diameter (in 16/26 splenomegalic patients; 1 pts. had been splenectomized and 9 were not splenomegalic at baseline). Toxicity was as expected, largely of grade (G) 1-2, always reversible and mainly consisting in: infusion reactions (G1-2 92%; G3 4%, in a single pt allergic to previous RTX who discontinued OBI); asymptomatic increase of liver enzymes (G1-2 19%; G3 GGT 15%; G3 ALT 4%); transient worsening of baseline G1-G3 thrombocytopenia to G3 (23%) or G4 (35%), with only 1 minor bleeding (4%, G1 epistaxis); transient neutropenia (G2 4%; G3 15%; G4 15%); and rare infections (G1-2 herpes labialis 8%; G3 pneumonia 4%). CR was reached in 12/25 evaluable pts (48%), including 2 pts with incomplete platelet recovery (~70000-90000/mmc) and 2 pts with delayed resolution of palpable splenomegaly. Notably, CR was achieved in 5/7 pts refractory to PA and 1/2 pts refractory to RTX. Furthermore, CR was negative for MRD in 9/12 pts (75%, including 3 pts with delayed MRD clearance), something never observed with vemurafenib. However, the kinetics of cytopenia resolution was slower compared to our previous vemurafenib trial, in that in pts obtaining CR with OBI neutrophil recovered to ≥1500/mmc after a median of 7 weeks (vs 4 weeks with vemurafenib) and platelets recovered to ≥100000/mmc after a median of 8 weeks (vs 2 weeks with vemurafenib). Prior exposure to RTX (n=12 pts) did not compromise OBI efficacy (5 CR, all MRD-negative, in 11 evaluable pts). In pts achieving CR with OBI, progression-free and MRD-free survival were both 100% at 56 months (range 20-66) and 48 months (range 6.5-60) of median follow-up, respectively (Fig.1). The 13 non-CR pts (minor response 7; no response 3; progression 3) were effectively salvaged with another course of OBI combined with vemurafenib, which will be presented at the meeting. CONCLUSIONS OBI frequently produced deep and durable responses in R/R HCL, to an extent apparently greater than RTX, thus qualifying as an active and safe chemotherapy-free strategy in this setting.

Vemurafenib and Obinutuzumab as Frontline Therapy for Hairy Cell Leukemia

BackgroundHairy cell leukemia (HCL) is characterized by the underlying genetic lesion of BRAFV600E and responsiveness to BRAF inhibitors. We assessed the safety and activity of the BRAF inhibitor vemurafenib combined with obinutuzumab in patients with previously untreated HCL.MethodsWe conducted a single-arm, multicenter clinical study of vemurafenib plus obinutuzumab. Vemurafenib 960 mg twice daily was administered for four cycles, and obinutuzumab was administered in cycles 2 to 4. The primary end point was complete remission (CR). Secondary end points included assessment of safety, minimal residual disease (MRD), and BRAF allele burden according to digital droplet polymerase chain reaction (ddPCR).ResultsThirty patients were enrolled in the study, and 27 patients completed all four cycles of treatments and achieved CR (90%; 95% confidence interval [CI], 73 to 98). Three patients discontinued the study early because of adverse events and were not evaluable for response. Of the 27 patients who achieved CR, 26 patients (96%; 95% CI, 81 to 99) achieved MRD negativity. BRAFV600E allele was undetectable by ddPCR in all 21 evaluable patients. At a median follow-up of 34.9 months (95% CI, 29.6 to 36.9), no patient experienced disease relapse. The most common vemurafenib-related adverse events were rash and arthralgia. Febrile neutropenia occurred in two patients, and blood or platelet transfusions were required in two patients.ConclusionsCombined time-limited vemurafenib and obinutuzumab achieved CR in more than 90% of patients with previously untreated HCL. In this small study, acquired vemurafenib resistance or dose-limiting toxicity was not observed. Patients were not observed long enough to reveal secondary malignancies. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT03410875.)

Somatic ARAF mutations in pediatric Langerhans cell histiocytosis: clinicopathologic, genetic and functional profiling.

ARAF mutations have been identified in a limited subset of patients with Langerhans cell histiocytosis (LCH), a rare disorder characterized by abnormal proliferation of Langerhans cells. LCH is primarily instigated by mutations in the mitogen-activated protein kinase (MAPK) signaling pathway, with BRAFV600E and MAP2K1 mutations constituting most cases. ARAF mutations in LCH highlight the heterogeneity of the disease and provide insights into its underlying molecular mechanisms. However, the occurrence of ARAF-positive LCH cases is extremely rare, with only two reported globally. Although they may be linked to a more aggressive form of LCH and a more severe clinical progression, the clinical significance and functional consequences of these mutations remain uncertain. We performed next-generation sequencing (NGS) to explore driver mutations in 148 pediatric LCH patients and recognized a series of mutations, including an identical novel somatic ARAF mutation, c.1046_1051delAGGCTT (p.Q349_F351delinsL), in four pediatric LCH patients. It was considered an ARAF hotspot mutation. All reported ARAF-positive patients worldwide exhibited characteristic pathological features of LCH, albeit with involvement across multiple systems. In vitro functional studies showed that this mutation could trigger the MAPKinase pathway and phosphorylate its downstream effectors MEK1/2 and ERK1/2 (relatively weaker than BRAFV600E). Over-activation of mutant A-Raf kinase could be inhibited by the BRAF inhibitor vemurafenib. LCH is uncommon, and ARAF mutation is even rarer. In our study, we have identified a novel hotspot somatic ARAF mutation, which has been verified through functional analysis to be an activating mutation. LCH patients with ARAF mutation typically have an unfavorable prognosis due to limited treatment experiences, although they do not exhibit a high relapse rate. To aid in the development of personalized treatment approaches and prognostic markers for LCH patients, it is recommended to conduct typical pathological and immunohistochemical examinations, as well as genetic tests utilizing a targeted gene panel or whole exome sequencing (WES), for LCH diagnosis, thereby promoting the use of inhibitor treatment strategies.