TGFß-Responsive Stromal Activation Occurs Early in Serrated Colorectal Carcinogenesis.

Activated TGFß signaling in the tumor microenvironment, which occurs independently of epithelial cancer cells, has emerged as a key driver of tumor progression in late-stage colorectal cancer (CRC). This study aimed to elucidate the contribution of TGFß-activated stroma to serrated carcinogenesis, representing approximately 25% of CRCs and often characterized by oncogenic BRAF mutations. We used a transcriptional signature developed based on TGFß-responsive, stroma-specific genes to infer TGFß-dependent stromal activation and conducted in silico analyses in 3 single-cell RNA-seq datasets from a total of 39 CRC samples and 12 bulk transcriptomic datasets consisting of 2014 CRC and 416 precursor samples, of which 33 were serrated lesions. Single-cell analyses validated that the signature was expressed specifically by stromal cells, effectively excluding transcriptional signals derived from epithelial cells. We found that the signature was upregulated during malignant transformation and cancer progression, and it was particularly enriched in CRCs with mutant BRAF compared to wild-type counterparts. Furthermore, across four independent precursor datasets, serrated lesions exhibited significantly higher levels of TGFß-responsive stromal activation compared to conventional adenomas. This large-scale analysis suggests that TGFß-dependent stromal activation occurs early in serrated carcinogenesis. Our study provides novel insights into the molecular mechanisms underlying CRC development via the serrated pathway.

Reconstitution and characterization of BRAF in complex with 14-3-3 and KRAS4B on nanodiscs.

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

Protein phosphatase 6 activates NF-κB to confer sensitivity to MAPK pathway inhibitors in KRAS- and BRAF-mutant cancer cells.

The Ras-mitogen-activated protein kinase (MAPK) pathway is a major target for cancer treatment. To better understand the genetic pathways that modulate cancer cell sensitivity to MAPK pathway inhibitors, we performed a CRISPR knockout screen with MAPK pathway inhibitors on a colorectal cancer (CRC) cell line carrying mutant KRAS. Genetic deletion of the catalytic subunit of protein phosphatase 6 (PP6), encoded by PPP6C, rendered KRAS- and BRAF-mutant CRC and BRAF-mutant melanoma cells more resistant to these inhibitors. In the absence of MAPK pathway inhibition, PPP6C deletion in CRC cells decreased cell proliferation in two-dimensional (2D) adherent cultures but accelerated the growth of tumor spheroids in 3D culture and tumor xenografts in vivo. PPP6C deletion enhanced the activation of nuclear factor κB (NF-κB) signaling in CRC and melanoma cells and circumvented the cell cycle arrest and decreased cyclin D1 abundance induced by MAPK pathway blockade in CRC cells. Inhibiting NF-κB activity by genetic and pharmacological means restored the sensitivity of PPP6C-deficient cells to MAPK pathway inhibition in CRC and melanoma cells in vitro and in CRC cells in vivo. Furthermore, a R264 point mutation in PPP6C conferred loss of function in CRC cells, phenocopying the enhanced NF-κB activation and resistance to MAPK pathway inhibition observed for PPP6C deletion. These findings demonstrate that PP6 constrains the growth of KRAS- and BRAF-mutant cancer cells, implicates the PP6-NF-κB axis as a modulator of MAPK pathway output, and presents a rationale for cotargeting the NF-κB pathway in PPP6C-mutant cancer cells.

Combined therapy of dabrafenib and an anti-HER2 antibody-drug conjugate for advanced BRAF-mutant melanoma.

BACKGROUND: Melanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma. METHODS: We evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy. RESULTS: Our results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway. CONCLUSION: These findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Treatment of Cerebral Histiocytosis With Low Dose of Cobimetinib: A Report of 2 Cases.

OBJECTIVES: Histiocytic disorders are pathologic expansions of myeloid cells in multiple organs, including the CNS. They share activation of the MAP kinase pathway due to either BRAFV600E variant or other variants in the RAS-RAF-MEK-ERK pathway. The rarity and heterogeneity of the disease only enable therapy through pathophysiologic considerations. METHODS: We present 2 histiocytosis cases without BRAF sequence variants that affect the CNS, one with Erdheim-Chester disease and the other with an unspecified histiocytosis, and their diagnostic and therapeutic challenges. RESULTS: In both cases, comprehensive analysis of the RAS-RAF-MEK-ERK signaling pathway secured the diagnosis. Treatment with the MEK inhibitor cobimetinib brought the disease to a complete halt. However, side effects such as thrombosis and serous macular edema made it necessary to reduce cobimetinib dosage. Low-dose cobimetinib maintenance medication was successful in preventing recurrence of histiocytic disease. DISCUSSION: CNS involvement of histiocytic disorders can lead to detrimental neurologic symptoms. MEK inhibitors are effective treatment options for some of these patients. Since side effects are common, according to our cases we propose a low-dose treatment of 20 mg per day to balance treatment effects with side effects. CLASSIFICATION OF EVIDENCE: This case report provides Class IV evidence. This is a single observational study without controls.

Genomic deletions explain the generation of alternative BRAF isoforms conferring resistance to MAPK inhibitors in melanoma.

Resistance to MAPK inhibitors (MAPKi), the main cause of relapse in BRAF-mutant melanoma, is associated with the production of alternative BRAF mRNA isoforms (altBRAFs) in up to 30% of patients receiving BRAF inhibitor monotherapy. These altBRAFs have been described as being generated by alternative pre-mRNA splicing, and splicing modulation has been proposed as a therapeutic strategy to overcome resistance. In contrast, we report that altBRAFs are generated through genomic deletions. Using different in vitro models of altBRAF-mediated melanoma resistance, we demonstrate the production of altBRAFs exclusively from the BRAF V600E allele, correlating with corresponding genomic deletions. Genomic deletions are also detected in tumor samples from melanoma and breast cancer patients expressing altBRAFs. Along with the identification of altBRAFs in BRAF wild-type and in MAPKi-naive melanoma samples, our results represent a major shift in our understanding of mechanisms leading to the generation of BRAF transcripts variants associated with resistance in melanoma.

Overcoming BRAF and CDK4/6 inhibitor resistance by inhibiting MAP3K3-dependent protection against YAP lysosomal degradation.

Transcriptional programs governed by YAP play key roles in conferring resistance to various molecular-targeted anticancer agents. Strategies aimed at inhibiting YAP activity have garnered substantial interest as a means to overcome drug resistance. However, despite extensive research into the canonical Hippo-YAP pathway, few clinical agents are currently available to counteract YAP-associated drug resistance. Here, we present a novel mechanism of YAP stability regulation by MAP3K3 that is independent of Hippo kinases. Furthermore, we identified MAP3K3 as a target for overcoming anticancer drug resistance. Depletion of MAP3K3 led to a substantial reduction in the YAP protein level in melanoma and breast cancer cells. Mass spectrometry analysis revealed that MAP3K3 phosphorylates YAP at serine 405. This MAP3K3-mediated phosphorylation event hindered the binding of the E3 ubiquitin ligase FBXW7 to YAP, thereby preventing its p62-mediated lysosomal degradation. Robust YAP activation was observed in CDK4/6 inhibitor-resistant luminal breast cancer cells. Knockdown or pharmacological inhibition of MAP3K3 effectively suppressed YAP activity and restored CDK4/6 inhibitor sensitivity. Similarly, elevated MAP3K3 expression supported the prosurvival activity of YAP in BRAF inhibitor-resistant melanoma cells. Inhibition of MAP3K3 decreased YAP-dependent cell proliferation and successfully restored BRAF inhibitor sensitivity. In conclusion, our study reveals a previously unrecognized mechanism for the regulation of YAP stability, suggesting MAP3K3 inhibition as a promising strategy for overcoming resistance to CDK4/6 and BRAF inhibitors in cancer treatment.

Targeting the EGFR/RAS/RAF signaling pathway in anticancer research: a recent update on inhibitor design and clinical trials (2020-2023).

INTRODUCTION: Recent years have seen significant strides in drug developmenttargeting the EGFR/RAS/RAF signaling pathway which is critical forcell growth and proliferation. Protein-protein interaction networksamong EGFR, RAS, and RAF proteins offer insights for drug discovery. This review discusses the drug design and development efforts ofinhibitors targeting these proteins over the past 3 years, detailingtheir structures, selectivity, efficacy, and combination therapy.Strategies to combat drug resistance and minimize toxicities areexplored, along with future research directions. AREA COVERED: This review encompasses clinical trials and patents on EGFR, KRAS,and BRAF inhibitors from 2020 to 2023, including advancements indesign and synthesis of proteolysis targeting chimeras (PROTACs) forprotein degradation. EXPERT OPINION: To tackle drug resistance, designing allosteric fourth-generationEGFR inhibitors is vital. Covalent, allosteric, or combinationaltherapies, along with PROTAC degraders, are key methods to addressresistance and toxicity in KRAS and BRAF inhibitors.

What Is the Timing and Role of Targeted Therapy in Metastatic Melanoma?

ABSTRACT: Melanoma is the most lethal cutaneous malignancy worldwide. The last 15 years have ushered in several regulatory approvals that have dramatically altered the landscape of treatment options for patients with melanoma. Many patients with melanoma harbor activating mutations in the BRAF proto-oncogene, a key component of the mitogen-activated protein kinase (MAPK) intracellular signaling pathway. Therapies targeting BRAF have led to remarkable improvements in both response rates and survival in patients with metastatic disease. In parallel with these developments in MAPK-targeted therapy has been the clinical development of immune checkpoint inhibitors, which also have improved response rates and survival in patients with metastatic disease including randomized trials compared with MAPK-targeted therapy in patients with advanced, BRAF-mutant melanoma. Immune checkpoint inhibitors have become the preferred first-line standard-of-care treatment for patients with newly diagnosed metastatic disease in patients irrespective of BRAF mutational status. Given these developments, it is now less clear how to optimize the use of MAPK-targeted therapy regarding treatment setting and in sequence with immune checkpoint inhibitor.

Anaplastic thyroid cancer spheroids as preclinical models to test therapeutics.

Anaplastic thyroid cancer (ATC) is the most aggressive thyroid cancer. Despite advances in tissue culture techniques, a robust model for ATC spheroid culture is yet to be developed. In this study, we created an efficient and cost-effective 3D tumor spheroids culture system from human ATC cells and existing cell lines that better mimic patient tumors and that can enhance our understanding of in vivo treatment response. We found that patient-derived ATC cells and cell lines can readily form spheroids in culture with a unique morphology, size, and cytoskeletal organization. We observed both cohesive (dense and solid structures) and discohesive (irregularly shaped structures) spheroids within the same culture condition across different cell lines. BRAFWT ATC spheroids grew in a cohesive pattern, while BRAFV600E-mutant ATC spheroids had a discohesive organization. In the patient-derived BRAFV600E-mutant ATC spheroids, we observed both growth patterns, but mostly the discohesive type. Histologically, ATC spheroids had a similar morphology to the patient's tumor through H&E staining and proliferation marker staining. Moreover, RNA sequencing analysis revealed that the gene expression profile of tumor cells derived from the spheroids closely matched parental patient tumor-derived cells in comparison to monolayer cultures. In addition, treatment response to combined BRAF and MEK inhibition in BRAFV600E-mutant ATC spheroids exhibited a similar sensitivity to the patient clinical response. Our study provides a robust and novel ex vivo spheroid model system that can be used in both established ATC cell lines and patient-derived tumor samples to better understand the biology of ATC and to test therapeutics.

Progress in long non-coding RNAs as prognostic factors of papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is generally recognized as a slow-growing tumor. However, a small subset of patients may still experience relapse or metastasis shortly after therapy, leading to a poor prognosis and raising concerns about excessive medical treatment. One major challenge lies in the inadequacy of effective biomarkers for accurate risk stratification. Long non-coding RNAs (lncRNAs), which are closely related to malignant characteristics and poor prognosis, play a significant role in the genesis and development of PTC through various pathways. The objective of this review is to provide a comprehensive summary of the biological functions of lncRNAs in PTC, identify prognosis-relevant lncRNAs, and explore their potential mechanisms in drug resistance to BRAF kinase inhibitors, tumor dedifferentiation, and lymph node metastasis. By doing so, this review aims to offer valuable references for both basic research and the prediction of PTC prognosis.

The MITF/mir-579-3p regulatory axis dictates BRAF-mutated melanoma cell fate in response to MAPK inhibitors.

Therapy of melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPKi) and immunotherapies. However, drug resistance continues to limit the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanomas. Among them, a pivotal role is played by the oncosuppressor miR-579-3p. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF, is able to stabilize MITF protein thus inducing its own transcription. From biological points of view, early exposure to MAPKi or, alternatively miR-579-3p transfection, induce block of proliferation and trigger senescence programs in BRAF-mutant melanoma cells. Finally, the long-term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to therapies in BRAF-mutant melanomas.

Effect of autologous dendritic cell cytokine-induced killer on refractory metastatic colorectal cancer: a matched case-control comparative study.

Background: Patients with metastatic colorectal cancer (mCRC) who are refractory to two or more lines of systemic chemotherapy have limited therapeutic options. The aim of this study was to evaluate the effect of autologous dendritic cell cytokine-induced killer (DC-CIK) transfer on the survival of patients with mCRC who are refractory or intolerant to at least two lines of systemic chemotherapies. Methods: A matched case-control comparative study was conducted with patients who received DC-CIK immunotherapy in addition to standard chemotherapy (cases) and those with standard chemotherapy alone (controls). The primary objective was to compare the duration of oncologic survival, including overall survival (OS) and progression-free survival (PFS), between the two groups. Results: A total of 27 cases and 27 controls were included. The median OS in the DC-CIK case group was 18.73 ± 5.48 months, which was significantly longer than that in the control group (14.23 ± 1.90 months, p = 0.045). However, there was no significant difference in PFS between the two groups (p = 0.086). Subgroup analysis showed that in patients with liver or extra-regional lymph node metastasis, DC-CIK cases had longer OS than controls (17.0 vs. 11.87 months, p = 0.019; not match vs. 6.93 months, p = 0.002, respectively). In patients with Eastern Cooperative Oncology Group (ECOG) scale 0 or wild RAS/BRAF, DC-CIK cases showed a significant increase in OS duration compared to controls (28.03 vs. 14.53 months, p = 0.038; 18.73 vs. 11.87 months, p = 0.013, respectively). Conclusions: The addition of autologous DC-CIK to standard chemotherapy had a positive effect on OS of patients with refractory mCRC, especially those with liver or extra-regional lymph node metastasis, ECOG = 0, and wild RAS/BRAF status.

Body size and risk of colorectal cancer molecular defined subtypes and pathways: Mendelian randomization analyses.

BACKGROUND: Obesity has been positively associated with most molecular subtypes of colorectal cancer (CRC); however, the magnitude and the causality of these associations is uncertain. METHODS: We used Mendelian randomization (MR) to examine potential causal relationships between body size traits (body mass index [BMI], waist circumference, and body fat percentage) with risks of Jass classification types and individual subtypes of CRC (microsatellite instability [MSI] status, CpG island methylator phenotype [CIMP] status, BRAF and KRAS mutations). Summary data on tumour markers were obtained from two genetic consortia (CCFR, GECCO). FINDINGS: A 1-standard deviation (SD:5.1 kg/m2) increment in BMI levels was found to increase risks of Jass type 1MSI-high,CIMP-high,BRAF-mutated,KRAS-wildtype (odds ratio [OR]: 2.14, 95% confidence interval [CI]: 1.46, 3.13; p-value = 9 × 10-5) and Jass type 2non-MSI-high,CIMP-high,BRAF-mutated,KRAS-wildtype CRC (OR: 2.20, 95% CI: 1.26, 3.86; p-value = 0.005). The magnitude of these associations was stronger compared with Jass type 4non-MSI-high,CIMP-low/negative,BRAF-wildtype,KRAS-wildtype CRC (p-differences: 0.03 and 0.04, respectively). A 1-SD (SD:13.4 cm) increment in waist circumference increased risk of Jass type 3non-MSI-high,CIMP-low/negative,BRAF-wildtype,KRAS-mutated (OR 1.73, 95% CI: 1.34, 2.25; p-value = 9 × 10-5) that was stronger compared with Jass type 4 CRC (p-difference: 0.03). A higher body fat percentage (SD:8.5%) increased risk of Jass type 1 CRC (OR: 2.59, 95% CI: 1.49, 4.48; p-value = 0.001), which was greater than Jass type 4 CRC (p-difference: 0.03). INTERPRETATION: Body size was more strongly linked to the serrated (Jass types 1 and 2) and alternate (Jass type 3) pathways of colorectal carcinogenesis in comparison to the traditional pathway (Jass type 4). FUNDING: Cancer Research UK, National Institute for Health Research, Medical Research Council, National Institutes of Health, National Cancer Institute, American Institute for Cancer Research, Brigham and Women's Hospital, Prevent Cancer Foundation, Victorian Cancer Agency, Swedish Research Council, Swedish Cancer Society, Region Västerbotten, Knut and Alice Wallenberg Foundation, Lion's Cancer Research Foundation, Insamlingsstiftelsen, Umeå University. Full funding details are provided in acknowledgements.

Cross-Species Comparison of the Pan-RAF Inhibitor LY3009120's Anti-Tumor Effects in Equine, Canine, and Human Malignant Melanoma Cell Lines.

Malignant melanomas (MMs) are the abnormal proliferation of melanocytes and are one of the lethal skin cancers in humans, equines, and canines. Accordingly, MMs in companion animals can serve as naturally occurring animal models, completing conventional cancer models. The common constitutive activation of the MAPK and PI3K pathways in MMs has been described in all three species. Targeting the related pathways is considered a potential option in comparative oncologic approaches. Herein, we present a cross-species comparative analysis exposing a set of ten melanoma cell lines (one human, three equine, and six canine) derived from primary tumors or metastasis to a pan-RAF and RAF dimer inhibitor (LY3009120). Cellular response (proliferation, biomass, metabolism, early and late apoptosis/necrosis, and morphology) and the presence of pathogenic single-nucleotide variants (SNVs) within the mutational hotspot genes BRAF exon 11 and 15, NRAS exon 2 and 3, KRAS exon 2, and KIT exon 11 were analyzed. This study showed that equine malignant melanoma (EMM) cells (MelDuWi) harbor the KRAS p.Q61H mutation, while canine malignant melanoma (CMM) cells (cRGO1 and cRGO1.2) carry NRAS p.G13R. Except for EMM metastasis cells eRGO6 (wild type of the above-mentioned hotspot genes), all melanoma cell lines exhibited a decrease in dose dependence after 48 and 72 h of exposure to LY3009120, independent of the mutation hotspot landscape. Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in all melanoma cell lines except for eRGO6. The anti-tumor effects of LY3009120 were observed in nine melanoma cell lines, indicating the potential feasibility of experimental trials with LY3009120. The present study reveals that the irradiation-resistant canine metastasis cells (cRGO1.2) harboring the NRAS p.G13R mutation are significantly LY3009120-sensitive, while the equine metastases-derived eRGO6 cells show significant resistance to LY3009120, which make them both valuable tools for studying resistance mechanisms in comparative oncology.

Killing capacity analysis of tumor-infiltrating cytotoxic lymphocytes and impact on lymph node metastasis in differentiated papillary carcinoma of thyroid with the BRAF V600E mutation.

BACKGROUND: Cytotoxic lymphocytes (CLs) express potent toxins, including perforin (P) and granzyme-B (G), which brings about target cell death. The purpose of this study was to evaluate the killing capacity of tumor-infiltrating CLs by means of P and G analysis, and explore the association with lymph node metastasis in papillary carcinoma of thyroid (PTC) without Hashimoto's thyroiditis (HT). METHODS: Infiltration of lymphocytes in PTC was observed in frozen sections. Both fresh tumor tissues and paracancerous tissues with lymphocyte infiltration were collected and prepared into a single cell suspension. Flow cytometry was used to detect the percentages of CD3+P+, CD3+G+, CD8+P+, and CD8+G+ T lymphocytes (TLs) and CD16-CD56+P+ and CD16-CD56+G+ natural killer (NK) cells. Finally, we investigated differential expression of P and G in NK cells and cytotoxic T lymphocytes (CTLs) in paired tumor tissues (group T, n = 44) and paracancerous tissues (group N, n = 44) from patients with PTC with the BRAF V600E mutation. Furthermore, patients were divided into two groups according to whether cervical central lymph node metastasis (CCLNM) existed: group A (with lymph node metastases, n = 27) and group B (with nonlymph node metastases, n = 17). Patients were also divided into three groups according to the total number of positive CCLNM: group B, group C (with low-level lymph node metastases, less than 5, n = 17) and group D (with high-level lymph node metastases, no less than 5, n = 10). RESULTS: The percentage of CD3+P+ CTLs was significantly higher in group N than in group T (P < 0.05). The percentage of CD8+G+ CTLs was significantly higher in group T than in group N (P < 0.05). The percentages of CD3+G+, CD16-CD56+P+and CD16-CD56+G+ NK cells showed no significant difference in either group T or group N (P > 0.05). The percentages of CD3+P+ CTLs in group A and group C were significantly higher in the paracancerous tissue than in the tumor tissue (P < 0.05). The percentages of CD8+G+ CTLs in group A and group C were significantly higher in the tumor tissues than in the paracancerous tissues (P < 0.05). The percentage of CD16-CD56+G+ NK cells in group D was significantly higher in the tumor tissues than in the paracancerous tissues (P < 0.05). CONCLUSIONS: The killing capacity of infiltrating CLs in PTC differed between tumor tissues and paracancerous tissues. In cases with CCLNM, higher expression of CD16-CD56+G+ NK cells in tumor tissues may be associated with a high risk of lymph node metastasis.

Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2.

The effect of targeted therapeutics on anticancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Because ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T-cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5, Mafg, and Zbtb7a. This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development toward monocytic lineage cells. In vivo, we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveal transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off-target effects of dabrafenib. SIGNIFICANCE: An important, but poorly understood, aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities.

Association of MDM2 Overexpression in Ameloblastomas with MDM2 Amplification and BRAFV600E Expression.

Ameloblastoma is a rare tumor but represents the most common odontogenic neoplasm. It is localized in the jaws and, although it is a benign, slow-growing tumor, it has an aggressive local behavior and high recurrence rate. Therefore, alternative treatment options or complementary to surgery have been evaluated, with the most promising one among them being a targeted therapy with the v-Raf murine sarcoma viral oncogene homologue B (BRAF), as in ameloblastoma the activating mutation V600E in BRAF is common. Studies in other tumors have shown that the synchronous inhibition of BRAF and human murine double minute 2 homologue (MDM2 or HDM2) protein is more effective than BRAF monotherapy, particularly in the presence of wild type p53 (WTp53). To investigate the MDM2 protein expression and gene amplification in ameloblastoma, in association with BRAFV600E and p53 expression. Forty-four cases of ameloblastoma fixed in 10% buffered formalin and embedded in paraffin were examined for MDM2 overexpression and BRAFV600E and p53 expression by immunohistochemistry, and for MDM2 ploidy with fluorescence in situ hybridization. Sixteen of forty-four (36.36%) cases of ameloblastoma showed MDM2 overexpression. Seven of sixteen MDM2-positive ameloblastomas (43.75%) were BRAFV600E positive and fifteen of sixteen MDM2-positive ameloblastomas (93.75%) were p53 negative. All MDM2 overexpressing tumors did not show copy number alterations for MDM2. Overexpression of MDM2 in ameloblastomas is not associated with MDM2 amplification, but most probably with MAPK activation and WTp53 expression. Further verification of those findings could form the basis for the use of MDM2 expression as a marker of MAPK activation in ameloblastomas and the trial of dual BRAF/MDM2 inhibition in the management of MDM2-overexpressing/BRAFV600E-positive/WTp53 ameloblastomas.

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.