MEK inhibition enhances presentation of targetable MHC-I tumor antigens in mutant melanomas

Combining multiple therapeutic strategies in NRAS/BRAF mutant melanoma, namely MEK/BRAF kinase inhibitors, immune checkpoint inhibitors, and targeted immunotherapies, may offer an improved survival benefit by overcoming limitations associated with any individual therapy. Still, optimal combination, order, and timing of administration remains under investigation. Here, we measure how MEK inhibition alters anti-tumor immunity by utilizing quantitative immunopeptidomics to profile changes the peptide MHC (pMHC) repertoire. These data reveal a collection of tumor antigens whose presentation levels are selectively augmented following therapy, including several epitopes present at over 1000 copies-per-cell. We leveraged the tunable abundance of MEKi-modulated antigens by targeting 4 epitopes with pMHC-specific T cell engagers and antibody drug conjugates, enhancing cell killing in tumor cells following MEK inhibition. These results highlight drug treatment as a means to enhance immunotherapy efficacy by targeting specific upregulated pMHCs and provide a methodological framework for identifying, quantifying, and therapeutically targeting additional epitopes of interest.

MEK Inhibition in a Newborn with RAF1-Associated Noonan Syndrome Ameliorates Hypertrophic Cardiomyopathy but Is Insufficient to Revert Pulmonary Vascular Disease

The RAF1:p.Ser257Leu variant is associated with severe Noonan syndrome (NS), progressive hypertrophic cardiomyopathy (HCM), and pulmonary hypertension. Trametinib, a MEK-inhibitor approved for treatment of RAS/MAPK-mutated cancers, is an emerging treatment option for HCM in NS. We report a patient with NS and HCM, treated with Trametinib and documented by global RNA sequencing before and during treatment to define transcriptional effects of MEK-inhibition. A preterm infant with HCM carrying the RAF1:p.Ser257Leu variant, rapidly developed severe congestive heart failure (CHF) unresponsive to standard treatments. Trametinib was introduced (0.022 mg/kg/day) with prompt clinical improvement and subsequent amelioration of HCM at ultrasound. The appearance of pulmonary artery aneurysm and pulmonary hypertension contributed to a rapid worsening after ventriculoperitoneal shunt device placement for posthemorrhagic hydrocephalus: she deceased for untreatable CHF at 3 months of age. Autopsy showed severe obstructive HCM, pulmonary artery dilation, disarrayed pulmonary vascular anatomy consistent with pulmonary capillary hemangiomatosis. Transcriptome across treatment, highlighted robust transcriptional changes induced by MEK-inhibition. Our findings highlight a previously unappreciated connection between pulmonary vascular disease and the severe outcome already reported in patients with RAF1-associated NS. While MEK-inhibition appears a promising therapeutic option for HCM in RASopathies, it appears insufficient to revert pulmonary hypertension.

SEMA6A/RhoA/YAP Axis Mediates Tumor-Stroma Interactions and Prevents Response to Dual BRAF/MEK Inhibition in BRAF-Mutant Melanoma

Background: Despite the promise of dual BRAF/MEK inhibition as a therapy for BRAF-mutant (BRAF-mut) melanoma, heterogeneous responses have been observed in patients, thus predictors of benefit from therapy are needed. We have previously identified semaphorin 6A (SEMA6A) as a BRAF-mut-associated protein involved in actin cytoskeleton remodeling. The purpose of the present study is to dissect the role of SEMA6A in the biology of BRAF-mut melanoma, and to explore its predictive potential towards dual BRAF/MEK inhibition.Methods: SEMA6A expression was assessed by immunohistochemistry in melanoma cohort RECI1 (N=112) and its prognostic potential was investigated in BRAF-mut melanoma patients from DFCI and TCGA datasets (N=258). The molecular mechanisms regulated by SEMA6A to sustain tumor aggressiveness and targeted therapy resistance were investigated in vitro by using BRAF-mut and BRAF-wt melanoma cell lines, an inducible SEMA6A silencing cell model and a microenvironment-mimicking fibroblasts-coculturing model. Finally, SEMA6A prediction of benefit from dual BRAF/MEK inhibition was investigated in melanoma cohort RECI2 (N=14).Results: Our results indicate higher protein expression of SEMA6A in BRAF-mut compared with BRAF-wt melanoma patients and show that SEMA6A is a prognostic indicator in BRAF-mut melanoma from TCGA and DFCI patients cohorts. In BRAF-mut melanoma cells, SEMA6A coordinates actin cytoskeleton remodeling by the RhoA-dependent activation of YAP and dual BRAF/MEK inhibition by dabrafenib+trametinib induces SEMA6A/RhoA/YAP axis. In microenvironment-mimicking co-culture condition, fibroblasts confer to melanoma cells a proliferative stimulus and protect them from targeted therapies, whereas SEMA6A depletion rescues the efficacy of dual BRAF/MEK inhibition. Finally, in BRAF-mut melanoma patients treated with dabrafenib+trametinib, high SEMA6A predicts shorter recurrence-free interval. Conclusions: Overall, our results indicate that SEMA6A contributes to microenvironment-coordinated evasion of melanoma cells from dual BRAF/MEK inhibition and it might be a good candidate predictor of short-term benefit from dual BRAF/MEK inhibition.

MEK Inhibition Demonstrates Activity in Relapsed, Refractory Patients with Juvenile Myelomonocytic Leukemia: Results from COG Study ADVL1521

Background: Juvenile myelomonocytic leukemia (JMML) is a hematologic malignancy of infants and toddlers with both myelodysplastic and myeloproliferative features. The prognosis for patients (pts) with relapsed or refractory (r/r) JMML is poor and hematopoietic stem cell transplant (HCT) is the only curative therapy. The molecular hallmark of JMML is activation of the Ras/MAPK pathway. In preclinical studies, MEK inhibition was shown to be effective at reducing spleen sizes, restoring normal hematopoiesis, and extending survival compared to placebo in several genetically engineered mouse models of JMML. Trametinib is an orally bioavailable MEK1/2 inhibitor and is approved for treatment of several malignancies in adults. This Children's Oncology Group study (ADVL1521, NCT03190915) is the first clinical trial for pts with r/r JMML conducted in the United States. Pts are eligible if they have persistent clinical or molecular evidence of JMML after 1 cycle of high dose cytarabine, 2 cycles of a hypomethylating agent or HCT. Pts receive daily trametinib for up to 12 cycles (28 days) in the absence of disease progression or dose-limiting toxicity (DLT). Dosing is age-based with pts less than 6 years of age receiving 0.032mg/kg/day and those 6 years or older receiving 0.025mg/kg/day. An oral suspension is available for pts unable to swallow tablets. Using a Simon 2-stage design (10 pts in each stage), trametinib would be deemed effective if 3 or more pts achieved an objective response. Results: From 2018-2021, 9 pts were enrolled; all 9 were eligible and evaluable for toxicity and response. Each pt had a detectable Ras mutation at the time of enrollment and was monitored for response using clinical and molecular criteria developed by an international consensus panel (Niemeyer et al, 2015). Five pts were less than 2 years of age. Three patients had relapsed post-HCT prior to enrolling and 6 patients were refractory to a median of 1.5 prior therapies (range 1-3). Four pts had an objective response (1 clinical complete response (cCR), 3 clinical partial responses, (cPR); 2 pts had stable disease and 3 had progressive disease (Table 1). Both pts with stable disease completed the maximum 12 cycles permitted on study. Two pts who achieved a cPR proceeded to HCT. One patient who achieved a cCR remains on study. No molecular responses were achieved. There were no dose-limiting toxicities; one pt had grade 4 thrombocytopenia probably related to trametinib. Of the 8 patients who consented to correlative studies, 7 had DNA methylation testing, 6 had kinome profiling, and 5 had RNASeq testing performed on both pre- and post-trametinib paired samples. DNA methylation testing revealed stable intrapatient methylation signatures across diagnostic, relapse and post-trametinib timepoints using the international consensus criteria (Schönung et al, 2020). Integrated kinome and RNASeq analysis revealed downregulation of proteins and genes involved in Ras/MAPK signaling. Conclusions: In the first clinical trial for r/r JMML patients in the United States, 4 objective responses were observed among the initial 9 patients meeting the pre-defined criteria to deem trametinib effective. While clinical responses including resolution of splenomegaly, resolution of monocytosis and increased platelets counts were observed, no molecular responses were noted. The treatment of r/r JMML has historically depended on HCT. Recently, azacitidine has shown promise in treating r/r JMML. This trial demonstrates that trametinib is active in r/r JMML and has a favorable side effect profile. Ongoing analysis of extensive correlative testing results have revealed potential mechanisms of response and resistance to MEK inhibition. Future studies will focus on children with newly diagnosed JMML and combine trametinib with azacitidine with or without HCT. Figure 1 Figure 1. Disclosures Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Barkauskas: Genentech: Current Employment.

Trametinib for Refractory Chylous Effusions in Children with Noonan Syndrome

BACKGROUND Noonan syndrome (NS) is one of several autosomal dominant multisystem disorders known as RASopathies. Common manifestations of NS include congenital heart defects and cardiomyopathy, lymphatic malformations, and predisposition to myeloproliferative disorders. Chylous fluid accumulation secondary to lymphatic malformations are seen in NS and are a major cause of morbidity and mortality often refractory to conventional medical management. There has been increasing interest in the use of pharmacologic MEK inhibition in the management of these patients given that activating RAS pathway mutations lead to downstream MEK activation that is causative of this pathology. DESIGN/METHODS Three patients with a confirmed diagnosis of NS are described. Each patient developed complications from chylous effusions refractory to conventional management and were subsequently enrolled on-study to treat with compassionate use oral trametinib from Novartis Pharmaceuticals on a single patient Investigational New Drug from the Food and Drug Administration (FDA). All patients were consented to be monitored for one year of therapy following a local protocol approved by the Colorado Institutional Review Board (COMIRB). Patient 1: a 4-year-old female with NS due to a pathogenic germline mutation of the RIT1 gene [c.246T>G, p.Phe82Leu] born with severe hypertrophic cardiomyopathy, mitral valve dysplasia, and pulmonary valve stenosis. She developed bilateral chylous pleural effusions that were refractory to dietary modification, diuretics, octreotide, and sirolimus. Patient 2: a 3-month-old female with NS due to a pathogenic germline mutation of the SOS1 gene [c.1322G>A; p.Cys441Tyr] born with esophageal atresia/tracheoesophageal fistula and moderate pulmonary valve stenosis. She developed bilateral chylous pleural effusions and ascites that were refractory to dietary modification and octreotide therapy. Patient 3: a 4-month-old male with NS due to a gain-of-function mutation of PTPN11 [c.854T>C; p.Phe285Ser] with hypertrophic cardiomyopathy, pulmonary valve stenosis, respiratory insufficiency with suspected pulmonary lymphangiectasia, and persistent chylous pleural effusions in addition to Noonan syndrome-associated myeloproliferative disorder (NS-MPD) that had been refractory to traditional management. RESULTS MEK inhibition with trametinib was used in three patients with NS and life-threatening complications with no medical or surgical treatment options. All three patients had dynamic contrast magnetic resonance lymphangiography (DCMRL) evidence of primary, central lymphatic dysplasia that manifested in lymphatic accumulation affecting cardiorespiratory function, nutrition, and the immune system. DCMRL imaging for patient 2 are highlighted in Figure 1 A and B. Within one month of initiating trametinib oral therapy, all three patients demonstrated response adequate to wean from mechanical ventilation and other supportive care modalities. Serum albumin levels improved as lymphatic leak resolved (Figure 1C). Patient 3 showed improvement in hypertrophic cardiomyopathy as evidenced by a decrease in both NT-proBNP and left ventricular mass by echocardiogram. Patients 1 and 2 demonstrated notable improvements in growth after one year of therapy, with increase in both weight and height percentiles. Patient 3 also presented with NS-MPD that responded with marked improvements in total WBC count as well as absolute monocyte count (Figure 1D). DISCUSSION Our experience adds to the growing body of evidence demonstrating the effectiveness of MEK inhibition on disease processes that are common in patients with NS and other RASopathies. None of the patients in our series experienced significant adverse effects from the medication aside from patient 2 who developed mild dermatitis. The efficacy of this therapy does not appear to be based on the underlying genotype, as each of the three patients we describe had different underlying molecular alterations (SOS1, RIT1, PTPN11). Substantial improvements in a variety of parameters including lymphatic malformations, cardiomyopathy, pulmonary valve stenosis, growth, and NS-MPD highlight the potential utility of trametinib in this patient population. Larger, prospective studies are necessary to confirm the efficacy of MEK inhibition and to assess the long-term safety of its use in this population. Figure 1 Figure 1. Disclosures Nakano: Novartis: Consultancy. OffLabel Disclosure: Trametinib is a MEK1/2 inhibitor that has been approved for the use in certain malignancies. Its off label use in children with Noonan Syndrome with significant lymphatic anomalies is based on the up regulation of the MAPK pathway in these patients.

CSIG-30. TARGETING TELOMERASE VIA MEK INHIBITION IN AGGRESSIVE TERT PROMOTER MUTATED BRAIN TUMORS

Activating point mutations within the TERT promoter (C228T or C250T) account for the most frequent alteration in aggressive brain tumors. Presence of these alterations results in the generation of binding sites for E-twenty-six (ETS) transcription factors accompanied by enhanced TERT expression. Accordingly, TERT promoter mutations foster cellular immortalization and subsequently tumor aggressiveness. Due to the limitation of treatment options in aggressive brain tumors, including glioblastoma and medulloblastoma, new therapeutic targets need to be discovered. As we previously described a strong interaction of oncogenic MEK/ETS signaling and TERT promoter mutations, we hypothesize that inhibition of these factors halters cell immortalization in TERT-driven brain tumors. Our study included three TERT promoter wild-type (TERTwt), six mutated (TERTmut) glioblastoma and three TERTmut medulloblastoma cell models and tested the effect of MEK inhibitors (U0126 and trametinib) and the ETS inhibitor YK-4-279 on cell viability and clone formation. Cellular senescence upon treatment was evaluated by beta-galactosidase assays. Impact on TERT mRNA expression and TERT promoter activity were analyzed by quantitative real-time PCR and luciferase reporter assays, respectively. Furthermore, the effects on MAPK- and PI3K pathway activation were evaluated by Western blot. Amongst the investigated inhibitors, tumor cells harboring C228T mutation were distinctly more sensitive against trametinib as compared to TERTwt and C250T TERTmut cells. Similar effects were observed on clonogenicity upon long-term exposure to this inhibitor. Regarding MAPK signaling activation, trametinib treatment completely blocked ERK phosphorylation in every cell model, while activation of ETS1 was more effectively reduced in C228T TERTmutcells. Accordingly, exposure to trametinib reduced TERT expression and promoter activity accompanied by induction of cellular senescence in cells with C228T mutation. Impact of trametinib is currently investigated in preclinical experiments using TERTmut brain tumor models. Summarizing, MEK inhibition represents a novel strategy to overcome cell immortalization especially in C228T TERTmut brain tumors.