Trametinib Treatment Research Articles

Trametinib Sensitivity is Defined by a Myeloid Differentiation Profile in Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML) is a common blood cancer marked by heterogeneity in disease and diverse genetic abnormalities. Additional therapies are needed as the 5-year survival remains below 30%. Trametinib is a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor that is widely used in solid tumors and also in tumors with activating RAS mutations. A subset of patients with AML carry activating RAS mutations; however, a small-scale clinical trial with trametinib showed little efficacy. Here, we sought to identify transcriptomic determinants of trametinib sensitivity in AML. We tested the activity of trametinib against a panel of tumor cells from patients with AML ex vivo and compared this with RNA sequencing (RNA-Seq) data from untreated blasts from the same patient samples. We then used a correlation analysis between gene expression and trametinib sensitivity to identify potential biomarkers predictive of drug response. We found that a subset of AML tumor cells were sensitive to trametinib ex vivo, only a fraction of which (3/10) carried RAS mutations. On the basis of our RNA-Seq analysis we found that markers of trametinib sensitivity are associated with a myeloid differentiation profile that includes high expression of CD14 and CLEC7A (Dectin-1), similar to the gene expression profile of monocytes. Further characterization confirmed that trametinib-sensitive samples display features of monocytic differentiation with high CD14 surface expression and were enriched for the M4 subtypes of the FAB classification. Our study identifies additional molecular markers that can be used with molecular features including RAS status to identify patients with AML that may benefit from trametinib treatment.

Preclinical evidence for employing MEK inhibition in NRAS mutated pediatric gastroenteropancreatic neuroendocrine-like tumors

BackgroundPediatric gastroenteropancreatic neuroendocrine tumors are exceedingly rare, resulting in most pediatric treatment recommendations being based on data derived from adults. Trametinib is a kinase inhibitor that targets MEK1/2 and has been employed in the treatment of cancers harboring mutations in the Ras pathway. MethodsWe utilized an established human pediatric gastroenteropancreatic neuroendocrine-like tumor patient-derived xenograft (PDX) with a known NRAS mutation to study the effects of MEK inhibition. We evaluated the effects of trametinib on proliferation, motility, and tumor growth in vivo. We created an intraperitoneal metastatic model of this PDX, characterized both the phenotype and the genotype of the metastatic PDX and again, investigated the effects of MEK inhibition. ResultsWe found target engagement with decreased ERK1/2 phosphorylation with trametinib treatment. Trametinib led to decreased in vitro cell growth and motility, and decreased tumor growth and increased animal survival in a murine flank tumor model. Finally, we demonstrated that trametinib was able to significantly decrease gastroenteropancreatic neuroendocrine intraperitoneal tumor metastasis. ConclusionsThe results of these studies support the further investigation of MEK inhibition in pediatric NRAS mutated solid tumors.

HGG-15. SYNERGISTIC EFFECTS OF COMBINED MEK/CDK4/6 INHIBITOR TREATMENT ON BRAFV600E POSITIVE HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric high-grade glioma (pHGG) is associated with poor overall survival and standard care has remained unchanged for the past decade. BRAFV600E mutations occur in 5-10% and dabrafenib in combination with mekinist is approved in this subtype. Nevertheless, acquired resistance to targeted therapeutics is a common setback in highly aggressive tumors. Homozygous CDKN2A/B loss co-occurs in approximately 60% of this pHGG subtype. Taking advantage of those combined molecular alterations and exploiting acquired resistance mechanisms is of utmost importance to increase overall survival in these patients. METHODS Four HGG cell models with BRAFV600E mutation and homozygous CDKN2A/B loss were tested for the effects of CDK4/6 inhibitor monotherapy (palbociclib, ribociclib, abemaciclib) and/or combination treatment with trametinib on cell survival, cell cycle, apoptosis and senescence. RESULTS Abemaciclib showed the highest efficacy of CDK4/6 inhibitors comparable to trametinib monotherapy in our cell models. Abemaciclib led to a cell cycle arrest and combined treatment with trametinib induced distinctly increased levels of apoptosis in our tumor models when compared to monotherapy and a great proportion of surviving cells were senescent. One cell model was orthotopically implanted in mice and abemaciclib and combined treatment with trametinib showed a slightly increased response when compared to trametinib alone. CONCLUSIONS Summarizing, treatment with abemaciclib showed promising therapeutic effects in HGG cell models with BRAFV600E mutation and homozygous CDKN2A/B loss comparable to trametinib alone. Levels of apoptosis were distinctly higher with combinatorial abemaciclib and trametinib treatment when compared to monotherapy alone and senescence was induced in a great number of surviving cells. In vivo abemaciclib was more efficient when compared to trametinib alone. Currently we are working on in vivo experiments and the effect on sustained induction of senescence and mechanisms of tumor cell growth after discontinuation of therapy and in a combinatorial metronomic approach and in combination with radiotherapy.

LGG-14. INTEGRATED MULTI-OMICS EXPLORATION OF MAPK PATHWAY INHIBITION IN PEDIATRIC PILOCYTIC ASTROCYTOMA

Abstract Pilocytic astrocytomas (PA), the most common pediatric brain tumors, exhibit MAPK pathway alterations leading to its constitutive activation. They are also associated with low proliferation index due to the oncogene-induced senescence (OIS), sustained by senescence-associated secretory phenotype (SASP) factors. Little is known about the downstream consequences of MAPK activation leading to OIS-SASP, and of the molecular implications of MAPK pathway inhibition in senescent PA cells. Senescent DKFZ-BT66 cells derived from a primary KIAA::BRAF-fusion positive PA were used to generate RNA-sequencing and phospho-/proteomic datasets, before and after treatment with the MEK inhibitor (MEKi) trametinib for different time-spans. A multi-omics factor analysis tool (MEFISTO) and single sample gene set enrichment analysis (ssGSEA) were used to identify key OIS effectors and differentially regulated pathways upon MAPK inhibition. Trametinib treatment inhibited OIS and SASP signatures across all omics levels in the senescent DKFZ-BT66 cells, associated with reduced sensitivity towards senolytics drugs, indicating inhibition of senescence features upon MEKi. MEFISTO analysis identified key effectors involved in MAPK-induced OIS in the senescent PA cells. We built a pathway network using a prior knowledge network approach allowing to map the direct downstream effectors of MAPK leading to OIS-SASP. We identified several parallel pathways (JAK/STAT, p38, GSK3B) potentially involved in OIS and under the control of the MAPK pathway. In addition, ssGSEA showed that pathways related to upstream MAPK activators (SOS1, CK2, several RTKs) were predicted to be upregulated upon treatment, highlighting putative targets to be further investigated. Our data suggest that MAPKi reverses OIS signaling in senescent PA cells, while inducing the activation of MAPK upstream regulators, identifying putative co-targets for the treatment of PA. Further validation of the targetability of these pathways is pending. Furthermore, the identification of the MAPK-related OIS-SASP genes provides insight into the regulation of OIS-SASP by the MAPK pathway.

LGG-39. REAL-LIFE STUDY OF THE EFFICACY AND TOLERANCE OF TRAMETINIB IN THE TREATMENT OF LOW-GRADE GLIOMAS IN CHILDREN WITH NEUROFIBROMATOSIS TYPE 1

Abstract BACKGROUND Low grade gliomas (LGG) are the most common brain tumors in children. Targeted therapies such as the MEK-inhibitor trametinib have revolutionized the management of pediatric LGGs. NF1 mutations generate misregulations in the RAS/MAP kinase pathway, but data on trametinib in type 1 neurofibromatosis (NF1)-related LGG are scarce. Efficacy and safety of trametinib in NF1 patients with LGG need to be further investigated. METHODS This retrospective study included patients with NF1 <18 years from two French centers having received trametinib for a relapsing/progressive LGG between March 2020 and July 2022. Treatment efficacy was assessed according to RAPNO criteria, and toxicities and therapeutic exposure were assessed using real-life data collected retrospectively up to July 2023. RESULTS The study enrolled 11 patients, the median age at LGG diagnosis was 3 (1-9) years. All patients previously received a median number of 2 (1-4) lines of chemotherapy. Median age at treatment initiation was 9 (4-13) years. Median duration of trametinib treatment was 9.3 (5.8-24) months. Progression-free survival was 91% at 6 months and 61% at 2 years. Median time to best response was 2.7 (1.1-9) months, including 5 stable diseases, 4 minor responses, and 2 partial responses. Median duration of response was 8.6 (3.7-36.2) months. Adverse events (AEs) occurred in all patients, and 64% of the patient had grade 3 AEs. Skin toxicities and paronychia were the most frequent AEs. Treatment interruption occurred in 55% of the patients, and 3 patients permanently discontinued trametinib due to treatment-related toxicities. Real therapeutic exposure was significantly shorter than theoretically expected (19.6% treatment reduction). CONCLUSION Trametinib showed promising efficacy in NF1 children with relapsing/progressive LGG. Trametinib toxicity remains challenging. Further prospective clinical trials stratified according to molecular alterations are needed to support these preliminary results.

Trametinib sensitizes KRAS-mutant lung adenocarcinoma tumors to PD-1/PD-L1 axis blockade via Id1 downregulation

BackgroundThe identification of novel therapeutic strategies to overcome resistance to the MEK inhibitor trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a challenge. This study analyzes the effects of trametinib on Id1 protein, a key factor involved in the KRAS oncogenic pathway, and investigates the role of Id1 in the acquired resistance to trametinib as well as the synergistic anticancer effect of trametinib combined with immunotherapy in KRAS-mutant LUAD.MethodsWe evaluated the effects of trametinib on KRAS-mutant LUAD by Western blot, RNA-seq and different syngeneic mouse models. Genetic modulation of Id1 expression was performed in KRAS-mutant LUAD cells by lentiviral or retroviral transductions of specific vectors. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis were measured by flow cytometry. The anti-tumor efficacy of the combined treatment with trametinib and PD-1 blockade was investigated in KRAS-mutant LUAD mouse models, and the effects on the tumor immune infiltrate were analyzed by flow cytometry and immunohistochemistry.ResultsWe found that trametinib activates the proteasome-ubiquitin system to downregulate Id1 in KRAS-mutant LUAD tumors. Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-mutant LUAD mouse models, we found that trametinib synergizes with PD-1/PD-L1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity depended on trametinib-mediated Id1 reduction and was associated with a less immunosuppressive tumor microenvironment and increased PD-L1 expression on tumor cells.ConclusionsOur data demonstrate that Id1 expression is involved in the resistance to trametinib and in the synergistic effect of trametinib with anti-PD-1 therapy in KRAS-mutant LUAD tumors. These findings suggest a potential therapeutic approach for immunotherapy-refractory KRAS-mutant lung cancers.Graphical

NOX4 promotes tumor progression through the MAPK-MEK1/2-ERK1/2 axis in colorectal cancer.

Metabolic reprogramming plays a key role in cancer progression and clinical outcomes; however, the patterns and primary regulators of metabolic reprogramming in colorectal cancer (CRC) are not well understood. To explore the role of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in promoting progression of CRC. We evaluated the expression and function of dysregulated and survival-related metabolic genes using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Consensus clustering was used to cluster CRC based on dysregulated metabolic genes. A prediction model was constructed based on survival-related metabolic genes. Sphere formation, migration, invasion, proliferation, apoptosis and clone formation was used to evaluate the biological function of NOX4 in CRC. mRNA sequencing was utilized to explore the alterations of gene expression NOX4 over-expression tumor cells. In vivo subcutaneous and lung metastasis mouse tumor model was used to explore the effect of NOX4 on tumor growth. We comprehensively analyzed 3341 metabolic genes in CRC and identified three clusters based on dysregulated metabolic genes. Among these genes, NOX4 was highly expressed in tumor tissues and correlated with worse survival. In vitro, NOX4 overexpression induced clone formation, migration, invasion, and stemness in CRC cells. Furthermore, RNA-sequencing analysis revealed that NOX4 overexpression activated the mitogen-activated protein kinase-MEK1/2-ERK1/2 signaling pathway. Trametinib, a MEK1/2 inhibitor, abolished the NOX4-mediated tumor progression. In vivo, NOX4 overexpression promoted subcutaneous tumor growth and lung metastasis, whereas trametinib treatment can reversed the metastasis. Our study comprehensively analyzed metabolic gene expression and highlighted the importance of NOX4 in promoting CRC metastasis, suggesting that trametinib could be a potential therapeutic drugs of CRC clinical therapy targeting NOX4.

Characterization of BRAFThr599dup Mutation as a Targetable Driver Mutation Identified in Lung Adenocarcinoma by Comprehensive Genomic Profiling.

Understanding the function of BRAF mutants is crucial for determining the best treatment strategy. This study aimed to characterize a rare BRAF variant, BRAFThr599dup, which was identified in a patient with lung adenocarcinoma (LUAD) by comprehensive genomic profiling. We report a case of LUAD with BRAFThr599dup treated with dabrafenib and trametinib. We conditionally expressed wild-type BRAF, BRAFV600E, or BRAFThr599dup in Ba/F3 cells and BEAS-2B cells. Ba/F3 cells carrying double-mutant BRAF (BRAFThr599dup/R509H, BRAFV600E/R509H, or BRAFK601E/R509H) that lacked the dimerizing ability were also established. Knockout of endogenous BRAF or CRAF in Ba/F3-BRAFThr599dup cells and Ba/F3-BRAFV600E cells was performed using the CRISPR/Cas9 system. Cell viability, mitogen-activated protein kinase (MAPK) signaling activity, and sensitivity to dabrafenib and trametinib were evaluated. The patient was revealed to have BRAFThr599dup-positive tumor cells as a predominant clone, and dabrafenib and trametinib treatment showed modest efficacy. In Ba/F3 cells, both BRAFThr599dup and BRAFV600E similarly caused interleukin-3-independent proliferation and activated the MAPK pathway. Moreover, BRAFThr599dup and BRAFV600E similarly caused a significant increase in the anchorage-independent growth ability of BEAS-2B cells. Along with Ba/F3-BRAFV600E cells, Ba/F3-BRAFThr599dup cells were highly sensitive to a monomer-specific BRAF inhibitor, dabrafenib, with a half-maximal inhibitory concentration value of 29.7 nM. In the absence of wild-type BRAF, wild-type CRAF, or an intact dimer interface, the ability to induce oncogenic addiction and MAPK pathway activation in Ba/F3-BRAFThr599dup cells was not affected, which was in contrast to the findings in the BRAFK601E/R509H double-mutant model. BRAFThr599dup is a potent driver oncogene that activates the MAPK pathway without the requirement for dimerization in vitro. Because BRAFThr599dup has been recurrently reported across various cancer types, our findings should be further investigated both mechanistically and clinically.

Abstract 4375: ALPPL2 is involved in MAPK signal pathway of pancreatic cancer

Abstract Background: Alkaline phosphatase placental-like 2 (ALPPL2) is a member of the alkaline phosphatase family. ALPPL2 is currently being investigated as a novel therapeutic target due to its cancer-specific expression. However, our knowledge of ALPPL2 biological function in cancer is limited. Here we describe an interaction between ALPPL2 and MAPK signaling pathway in pancreatic cancer. Materials and Methods: ALPPL2 mRNA expression in a panel of 27 pancreatic cell lines was determined by RNAseq. Protein expression and phosphorylation were measured by Western blot. ALPPL2 or ALPP were overexpressed in pancreas cell lines through a Lentiviral packaging system. MEK inhibitor (trametinib) activity was assessed in the same panel of cell lines using a 6-day cell proliferation assay on a Synentec Cellavista imaging system. MEK inhibitor resistant cell lines were developed by culturing cells in increasing concentrations of MEK162. Results: We hypothesized that pancreatic cancer cells with high ALPPL2 expression may be less sensitive to MEK inhibition. Our study from a panel of 27 pancreatic cancer cell lines showed that there was no correlation between ALPPL2 expression and response to trametinib. Western blot showed that the level of phospho-ERK was reduced in PANC1 and HPAC cells at 6 hours after trametinib treatment. Signaling rebounded at 24 and 48 hours, though it remained below baseline. Surprisingly, ALPPL2 expression increased at 24 and 48 hours after trametinib treatment, however, the mRNA of ALPPL2 and ALPP was down-regulated in three pancreatic cancer cell lines conditioned to be MEK inhibitor resistant. To understand the role of ALPPL2 in the MAPK signaling pathway in response to MEK inhibition, ALPPL2 and ALPP were overexpressed in 2 pancreatic cancer cell lines: YAPC and PSN-1 whose ALPPL2 and ALPP were undetected by Western blot prior to transfection. Overexpression of ALPPL2 or ALPP in YAPC cells resulted in more ERK phosphorylation with no change in total ERK, it also resulted in increased total and phospho-S6, and decreased phospho-STAT3 with no change in total STAT3. The amount of total and phospho-ERK were not changed in PSN-1 with overexpression of ALPPL2 or ALPP, however, it did result in increased total and phospho-S6, decreased phospho-STAT3. Interestingly, the expression of ALPPL2 and ALPP in YAPC cells with ALPPL2 or ALPP overexpression was increased at 48 hours after trametinib treatment. Discussion: To our knowledge, this is the first study to report that ALPPL2 interacts with the MAPK signaling pathway in pancreatic cancer cells. The activated MAPK signaling pathway appears to control ALPPL2 expression. The MAPK signaling pathway may regulate ALPPL2 expression through transcription and protein metabolism. We have also shown that ALPPL2 expression can activate MAPK signaling pathway. Our findings that ALPPL2 expression can be increased after trametinib treatment provide further rationale for targeting ALPPL2 in cancer. Citation Format: Ke Wei Gong, Bilal Hamid, Kenny W. Castro, Martina S. McDermott, Forrest Epstein, Kevin Chau, Chuhong Hu, Jun Zhang, Ming Lu, Benjamin G. Hoffstrom, Neil A. O'Brien, Dennis J. Slamon. ALPPL2 is involved in MAPK signal pathway of pancreatic cancer [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 4375.

Abstract A026: Identifying mechanisms to target in combination with trametinib to improve therapeutic efficacy in low-grade serous ovarian cancer

Abstract A026: Identifying mechanisms to target in combination with trametinib to improve therapeutic efficacy in low-grade serous ovarian cancer

Combined BET and MEK Inhibition synergistically suppresses melanoma by targeting YAP1.

Rationale: The response rate to the MEK inhibitor trametinib in BRAF-mutated melanoma patients is less than 30%, and drug resistance develops rapidly, but the mechanism is still unclear. Yes1-associated transcriptional regulator (YAP1) is highly expressed in melanoma and may be related to MEK inhibitor resistance. The purpose of this study was to investigate the mechanism of YAP1 in MEK inhibitor resistance in melanoma and to screen YAP1 inhibitors to further determine whether YAP1 inhibition reverses MEK inhibitor resistance. Methods: On the one hand, we analyzed paired melanoma and adjacent tissue samples using RNA-seq and found that the Hippo-YAP1 signaling pathway was the top upregulated pathway. On the other hand, we evaluated the transcriptomes of melanoma samples from patients before and after trametinib treatment and investigated the correlation between YAP1 expression and trametinib resistance. Then, we screened for inhibitors that repress YAP1 expression and investigated the mechanisms. Finally, we investigated the antitumor effect of YAP1 inhibition combined with MEK inhibition both in vitro and in vivo. Results: We found that YAP1 expression levels upon trametinib treatment in melanoma patients were correlated with resistance to trametinib. YAP1 was translocated into the nucleus after trametinib treatment in melanoma cells, which could render resistance to MEK inhibition. Thus, we screened for inhibitors that repress YAP1 expression and identified multiple bromodomain and extra-terminal (BET) inhibitors, including NHWD-870, as hits. BET inhibition repressed YAP1 expression by decreasing BRD4 binding to the YAP1 promoter. Consistently, YAP1 overexpression was sufficient to reverse the proliferation defect caused by BRD4 depletion. In addition, the BET inhibitor NHWD-870 acted synergistically with trametinib to suppress melanoma growth in vitro and in vivo. Conclusions: We identified a new vulnerability for MEK inhibitor-resistant melanomas, which activated Hippo pathway due to elevated YAP1 activity. Inhibition of BRD4 using BET inhibitors suppressed YAP1 expression and led to blunted melanoma growth when combined with treatment with the MEK inhibitor trametinib.

Abstract C010: A comprehensive predictive biomarker analysis for MEK Inhibitors in Patient-derived Xenograft (PDX) models

Abstract Introduction: The RAS-RAF-MEK-ERK pathway has been extensively studied for its prominent role in cancer development. Activating mutations in receptor tyrosine kinases, RAS, or RAF, are commonly observed in cancer. Pathway activation caused by these mutations is mediated through MEK kinases, making MEK inhibition an attractive therapeutic strategy. Although several in vitro studies have been carried out to identify predictive biomarkers for sensitivity to MEK inhibition, the results have not been validated in vivo. Methods: Cryopreserved or fresh PDX tumor tissues (~2–3 mm in diameter) were transplanted into NOD/SCID or BALB/c nude mice subcutaneously. Trametinib was administered orally daily at 0.3mg - 1mg/kg of body weight, starting when tumor size reached 100-300 mm3. Sequencing reads were aligned to both human and mouse reference genomes. Based on which alignment yielded fewer mismatches, they were subsequently sorted into human or mouse groups, representing cancer and stromal transcriptomes, respectively. Ambiguous readings were discarded. Transcript abundance was quantified using the Kallisto software tool. Nucleotide variants in human genes were detected using the GATK best practice workflow. Results: We systematically analyzed tumor growth data collected from 62 PDX models in 9 cancer types (colorectal, head and neck, esophageal, kidney, melanoma, lung, gastric, ovarian, and pancreatic) treated with Trametinib, an allosteric MEK1/2 inhibitor. The responses to Trametinib were classified into 3 categories based on the amount of tumor growth inhibition (TGI) achieved. About 20% of these models were sensitive to Trametinib treatment (TGI > 70% compared to vehicle), 35% were resistant (TGI ≤ 30% compared to vehicle), and the remaining showed a partial response (30% < TGI ≤ 70% compared to vehicle). Cross-group comparisons of PDX transcriptomic and mutation profiles allowed identification of novel biomarkers as well as validation of biomarkers previously reported in in vitro studies. Conclusions: The different sensitivity to MEK inhibition observed in PDX models mirrors the patient heterogeneity observed in clinic. Thus, our results highlight the need to identify the appropriate genetic background using predictive biomarkers when applying targeted therapies. Citation Format: Yanghui Sheng, Wubin Qian, Jingjing Wang, Sheng Guo. A comprehensive predictive biomarker analysis for MEK Inhibitors in Patient-derived Xenograft (PDX) models [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 C010.

Unraveling Clonal Evolution and Mechanisms of Treatment Resistance in Murine KMT2A-Rearranged Leukemia with Subclonal FLT3 N676K

Our understanding of how individual mutations, whether present in all or just a subset of the leukemia cells, affect cellular response to therapy is limited. Herein, we investigate the impact of different therapies on survival, evolution, and resistance patterns in an acute myeloid leukemia (AML) mouse model driven by KMT2A::MLLT3 and subclonal FLT3 N676K. Leukemic bone marrow expressing KMT2A::MLLT3-mCherry in all cells and FLT3 N676K-GFP in 40% of cells was re-transplanted into sublethally irradiated recipients. Upon engraftment, recipients were subjected to different treatments, including chemotherapy (cytarabine for 5 days+doxorubicin for 3 days), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Targeted treatment was administered for 28 days, while the control group received vehicle ( Fig 1a). Each treatment prolonged survival with a median latency of 30 days for chemotherapy 37.5 days for AC220, 42 days for chemotherapy+AC220, and 45 days for AC220+Trametenib, and 25.5 days for control mice. At sacrifice, all mice displayed splenomegaly and leukocytosis. The treatment impacted the evolution of the KMT2A:: MLLT3+ FLT3 N676K cells and they expanded to clonal dominance in control and chemotherapy-treated mice. However, three distinct patterns of evolution were detected in AC220-treated mice with the FLT3 N676K-containing cells either expanding to clonal dominance (>80% of KMT2A:: MLLT3+ FLT3 N676K), the FLT3 N676K-containing cells being eradicated (>80% of KMT2A::MLLT3-alone), or dual similar sized clones expressing KMT2A:: MLLT3-alone or KMT2A::MLLT3+ FLT3 N676K(Fig1 b).Eradication of the FLT3 N676K-cells was rare, predominantly observed upon AC220+Trametinib treatment and the frequency of dual clones increased when chemotherapy was followed by AC220, in line with treatment affecting the evolution of genetically distinct clones. RNA-sequencing was used to determine altered gene expression patterns upon resistance and unveiled three major clusters including 1) mice expressing KMT2A:: MLLT3-alone, 2) control and chemotherapy-treated mice, and 3) AC220-treated mice. Notably, a subset of AC220-treated mice clustered close to the control and chemotherapy-treated mice suggesting potential resistance mechanisms through activation of the MEK/ERK-pathway. All leukemias had a myeloid Cd11b + phenotype. However, the control and chemotherapy-treated leukemias also displayed an aberrant expression of the lymphoid marker B220 and showed enrichment of gene sets correlating with stemness and oxidative phosphorylation, that suggested a switch in cellular phenotype and metabolic state. In contrast, AC220-treated leukemias lacked or had a low fraction of B220 + cells and showed enrichment of gene sets correlating with granulocyte-monocyte progenitors (GMP) and immune regulatory pathways (Fig 1 c). Acquired AC220-resistance mutations was rare (n=2), suggesting activation of alternative pathways upon resistance. Re-treatment of established cell lines from treated animals with AC220 showed that the cells were resistant. To uncover mechanisms of resistance at the cellular level, single cell RNA-sequencing was performed on four mice and revealed the emergence of distinct cellular subpopulations in AC220-treated mice, with a strong GMP signature and upregulation of the homeobox gene Six1(Fig 1d).Overexpression of Six1 in all AC220-treated leukemias was further confirmed by bulk RNA-sequencing. Interestingly, Six1 has previously been described as a regulator for leukemia stem cell maintenance in AML and under Wnt/ß-catenin signalling control. Subsequent drug assays showed AC220-treated cells exhibited sensitivity to Six1 pharmacological inhibition, suggesting a dependency on Six1, as well as sensitivity to Wnt/ß-catenin inhibition. Taken together, the treatment given affected survival and impacted evolution of the FLT3 N676K-leukemia cells. The general absence of acquired mutations in most mice suggests that target-independent mechanisms may underline acquired resistance in most mice, and we propose the Six1/Wnt/ß-catenin axis as a potential vulnerability upon AC220-resistance.

Trametinib Maintenance in Patients with RAS Mutated Hematologic Malignancy Undergoing Allogeneic Haematopoietic Stem-Cell Transplantation: A Retrospective Transplant Study

Aim: The abnormal activation of MAPK signaling pathway plays an important role in the occurrence and development of leukemia. Whether MEK inhibitors can effectively reduce the recurrence of hematologic malignancy, We reviewed the efficacy and safety of trametinib maintenance in post-transplant patients with RAS mutations. Methods: Our study retrospectively analyzed 61 hematologic malignities with RAS mutation who underwent allo-HSCT at Beijing Gaobo Boren Hospital between January 2018 and January 2023. Twenty-five patients received maintenance therapy with trametinib post-HSCT(trametinib group), and 36 patients did not receive any maintenance therapy (control group). Trametinib group and control group also achieved CR at 2 months post-HSCT. Results: The diagnosed including AML (23, 37.7%), B-ALL (33, 54.1%), T-ALL (3, 5.0%), and 2 others (3.3%). Male: female = 29:32, The median age was 15 (1-68) years old. Before transplantation, 5 patients (8.2%) had extramedullary lesions and 16 patients (26.2%) had chromosomal abnormalities. The disease status before transplant was CR (42,68.9%),PR(6,9.7%),NR (13,21.3%) . 53 patients (86.9%) received second-line treatment. Fifteen cases (24.6%) were secondary transplants. Donor type included haploidential (45, 73.8%), identical sibling (5, 8.2%), and unrelated (11, 18.0%). Myeloablative conditioning regimen with eitherTBI/fludarabine (33, 54.1%) based or busulfan/fludarabine (28, 45.9%) based were applied. There were no significant differences between the two groups in age, sex, extramedullary disease,central nervous leukemia, karyotype analysis, disease status, conditioning regimen, donor type,acute and chronic GVHD and acute GVHD grades. The median follow-up time was 16.5 months (95%CI: 16.8-24.8 months), and the overall survival(OS) was 81.2% (95%CI: 68.6-89.1%). The disease-free survival (PFS) was 73.0% (95%CI: 59.7-82.5%). Trametinib group, the OS was 91.5% (95%CI: 70.0-97.8%), PFS was 88.0% (95%CI: 67.3-96.0%). Control group, the OS was 73.9% (95%CI: 55.9-85.6%), PFS was 65.8% (95%CI: 47.7-78.9%). Trametinib significantly improved the OS and DFS (P=0.044; P=0.041). In the trametinib group, 4 patients died after treatment, of which 3 died of relapse and 1 died of respiratory failure. In the non-trametinib group, 13 patients died, of which 6 died of relapse and 7 died of infection, stroke, GVHD and other causes. Trametinib treatment was initiated at a median of 94 days (range 63-178) at a median dose of 1 (range 0.5-2) mg daily.Trametinib was temporarily discontinued in 9 patients and dose modification in 12 patients because of side effects. mainly due to side effects including hematotoxicity in 5 patients, nosebleed in 4 patients, diarrhea in 1 patient, fatigue in 1 patient, and disease recurrence in 1 patient. The median modified daily dose was 0.5mg (range 0-1mg). Grade 2 AES occurred during the course of trametinib treatment, and no ≥ grade 3 AES was observed. The most common adverse events were neutropenia, anemia, thrombocytopenia, epistaxis, neutropenic fever, and fatigue, all of which were tolerable and reversible. No cardiorespiratory, renal, or neurotoxicity, or treatment-related deaths were observed. The median duration of trametinib maintenance therapy was 270 days (range 30-630 days). Conclusions: In this study, We have observed that trametinib maintenance post-transplantation can significantly improve the prognosis of patients with RAS mutations.Trametinib can be used as maintenance therapy with different conditioning regiments post-HSCT, which does not increase the risk of GVHD and organ injury, and is safe and effective. Future prospective clinical trials with larger sample sizes are needed for further research.

A real-world analysis of trametinib in combination with hydroxychloroquine or CDK4/6 inhibitor as third- or later-line therapy in metastatic pancreatic adenocarcinoma

BackgroundThere are no standard third-line treatment options for metastatic pancreatic ductal adenocarcinoma (mPDAC). Trametinib in combination with hydroxychloroquine (HCQ) or CDK4/6 inhibitors for pancreatic adenocarcinoma showed promising efficacy in preclinical studies. However, the regimens have not been well examined in patients with mPDAC.MethodsPatients with mPDAC who received the combination of trametinib and HCQ or CDK4/6 inhibitors as third- or later-line therapy were reviewed. The efficacy and prognosis were further analyzed.ResultsA total of 13 mPDAC patients were enrolled, of whom 8 and 5 patients were treated with trametinib plus HCQ or a CDK4/6 inhibitor (palbociclib or abemaciclib), respectively. All enrolled patients had either KRAS G12D or G12V mutations and had received a median of 3 prior lines of therapy (range, 2–6). The median trametinib treatment duration was 1.4 months. Of the 10 patients with measurable disease, only 1 patient achieved stable disease, and the remaining patients had progressive disease. Moreover, in patients treated with trametinib plus HCQ and a CDK4/6 inhibitor, the median progression-free survival was 2.0 and 2.8 months, respectively, and the median overall survival was 4.2 and 4.7 months, respectively. Moreover, 5 (50%) patients experienced grade 3–4 adverse events in 10 patients with available safety data.ConclusionsThe combination of trametinib and HCQ or CDK4/6 inhibitors may not be an effective later-line treatment for mPDAC, and the current preliminary findings need to be confirmed by other studies with larger sample sizes.

Senescence, regulators of alternative splicing and effects of trametinib treatment in progeroid syndromes

Progeroid syndromes such as Hutchinson Gilford Progeroid syndrome (HGPS), Werner syndrome (WS) and Cockayne syndrome (CS), result in severely reduced lifespans and premature ageing. Normal senescent cells show splicing factor dysregulation, which has not yet been investigated in syndromic senescent cells. We sought to investigate the senescence characteristics and splicing factor expression profiles of progeroid dermal fibroblasts. Natural cellular senescence can be reversed by application of the senomorphic drug, trametinib, so we also investigated its ability to reverse senescence characteristics in syndromic cells. We found that progeroid cultures had a higher senescence burden, but did not always have differences in levels of proliferation, DNA damage repair and apoptosis. Splicing factor gene expression appeared dysregulated across the three syndromes. 10 µM trametinib reduced senescent cell load and affected other aspects of the senescence phenotype (including splicing factor expression) in HGPS and Cockayne syndromes. Werner syndrome cells did not demonstrate changes in in senescence following treatment. Splicing factor dysregulation in progeroid cells provides further evidence to support this mechanism as a hallmark of cellular ageing and highlights the use of progeroid syndrome cells in the research of ageing and age-related disease. This study suggests that senomorphic drugs such as trametinib could be a useful adjunct to therapy for progeroid diseases.

Cross-species analysis of SHH medulloblastoma models reveals significant inhibitory effects of trametinib on tumor progression

Sonic Hedgehog (SHH) medulloblastomas (MBs) exhibit an intermediate prognosis and extensive intertumoral heterogeneity. While SHH pathway antagonists are effective in post-pubertal patients, younger patients exhibit significant side effects, and tumors that harbor mutations in downstream SHH pathway genes will be drug resistant. Thus, novel targeted therapies are needed. Here, we performed preclinical testing of the potent MEK inhibitor (MEKi) trametinib on tumor properties across 2 human and 3 mouse SHH MB models in vitro and in 3 orthotopic MB xenograft models in vivo. Trametinib significantly reduces tumorsphere size, stem/progenitor cell proliferation, viability, and migration. RNA-sequencing on human and mouse trametinib treated cells corroborated these findings with decreased expression of cell cycle, stem cell pathways and SHH-pathway related genes concomitant with increases in genes associated with cell death and ciliopathies. Importantly, trametinib also decreases tumor growth and increases survival in vivo. Cell cycle related E2F target gene sets are significantly enriched for genes that are commonly downregulated in both trametinib treated tumorspheres and primary xenografts. However, IL6/JAK STAT3 and TNFα/NFκB signaling gene sets are specifically upregulated following trametinib treatment in vivo indicative of compensatory molecular changes following long-term MEK inhibition. Our study reveals a novel role for trametinib in effectively attenuating SHH MB tumor progression and warrants further investigation of this potent MEK1/2 inhibitor either alone or in combination with other targeted therapies for the treatment of SHH MB exhibiting elevated MAPK pathway activity.

Activation of the EGFR/PI3K/AKT pathway limits the efficacy of trametinib treatment in head and neck cancer.

Blocking the mitogen-activated protein kinase (MAPK) pathway with the MEK1/2 inhibitor trametinib has produced promising results in patients with head and neck squamous cell carcinoma (HNSCC). In the current study, we showed that trametinib treatment leads to overexpression and activation of the epidermal growth factor receptor (EGFR) in HNSCC cell lines and patient-derived xenografts. Knockdown of EGFR improved trametinib treatment efficacy both in vitro and in vivo. Mechanistically, we demonstrated that trametinib-induced EGFR overexpression hyperactivates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. In vitro, blocking the PI3K pathway with GDC-0941 (pictilisib), or BYL719 (alpelisib), prevented AKT pathway hyperactivation and enhanced the efficacy of trametinib in a synergistic manner. In vivo, a combination of trametinib and BYL719 showed superior antitumor efficacy vs. the single agents, leading to tumor growth arrest. We confirmed our findings in a syngeneic murine head and neck cancer cell line in vitro and in vivo. Taken together, our findings show that trametinib treatment induces hyperactivation of EGFR/PI3K/AKT; thus, blocking of the EGFR/PI3K pathway is required to improve trametinib efficacy in HNSCC.

Direct early growth response-1 knockdown decreases melanoma viability independent of mitogen-activated extracellular signal-related kinase inhibition.

To investigate downstream molecular changes caused by mitogen-activated protein kinase (MEK) inhibitor treatment and further explore the impact of direct knockdown of early growth response-1 ( EGR1 ) in melanoma cell culture. RNA-sequencing (RNA-Seq) was performed to determine gene expression changes with MEK inhibitor treatment. Treatment with MEK inhibitor (trametinib) was then assessed in two cutaneous (MEL888, MEL624) and one conjunctival (YUARGE 13-3064) melanoma cell line. Direct knockdown of EGR1 was accomplished using lentiviral vectors containing shRNA. Cell viability was measured using PrestoBlueHS Cell Viability Reagent. Total RNA and protein were assessed by qPCR and SimpleWestern. RNA-Seq demonstrated a profound reduction in EGR1 with MEK inhibitor treatment, prompting further study of melanoma cell lines. Following trametinib treatment of melanoma cells, viability was reduced in both cutaneous (MEL888 26%, P < 0.01; MEL624 27%, P < 0.001) and conjunctival (YUARGE 13-3064 33%, P < 0.01) melanoma compared with DMSO control, with confirmed EGR1 knockdown to 0.04-, 0.01-, and 0.16-fold DMSO-treated levels (all P < 0.05) in MEL888, MEL624, and YUARGE 13-3064, respectively. Targeted EGR1 knockdown using shRNA reduced viability in both cutaneous (MEL624 78%, P = 0.05) and conjunctival melanoma (YUARGE-13-3064 67%, P = 0.02). RNA-Sequencing in MEK inhibitor-treated cells identified EGR1 as a candidate effector molecule of interest. In a malignant melanoma cell population, MEK inhibition reduced viability in both cutaneous and conjunctival melanoma with a profound downstream reduction in EGR1 expression. Targeted knockdown of EGR1 reduced both cutaneous and conjunctival melanoma cell viability independent of MEK inhibition, suggesting a key role for EGR1 in melanoma pathobiology.

Dabrafenib and trametinib treatment in patients with braf-mutated advanced thyroid cancer

Dabrafenib and trametinib treatment in patients with braf-mutated advanced thyroid cancer