Single Agent MEK Inhibition Research Articles

Targeting RAF1 gene fusions with MEK inhibition in metastatic melanoma

Abstract The biological and clinical relevance of gene fusions in melanoma is unknown. Reports and preclinical data have suggested that tumor cells with specific rearrangements such as RAF1 gene fusions could be therapeutically targeted. To investigate the relevance of targeted therapy in patients with melanoma harboring RAF1 gene fusions, we reviewed records of 1268 melanoma patients with targeted sequencing data at the Dana-Farber Cancer Institute. We identified 9 cases and report here on their clinicopathologic characteristics. We describe the favorable outcome of 2 patients who received MEK inhibitor therapy, including 1 patient with a durable response. We coalesced our data with published reports of patients with RAF1 gene fusions who were treated with targeted therapy. We find that single-agent MEK inhibition has anti-tumor activity in melanoma patients harboring an RAF1 gene fusion, and we propose that patients with RAF1 gene fusions should be considered for single-agent MEK inhibitor therapy.

LGG-02. Cardiac toxicity in patients receiving single-agent MEK inhibition

BACKGROUND: MEK inhibitor therapy is increasingly being utilized for the treatment of pediatric tumors, including low-grade glioma, plexiform neurofibroma and Langerhans cell histiocytosis. These drugs are well-tolerated but do have risk of toxicity, including cardiac toxicity. The purpose of this study is to better characterize MEK inhibitor-induced cardiac toxicity in pediatric patients. METHODS: Retrospective review of all patients who underwent MEK inhibitor mono-therapy for at least 3 months, 2015- 2021, age 25 years or less, at St. Louis Children’s hospital and Cardinal Glennon Children's hospital. RESULTS: We evaluated 31 patients, 19 (61%) with brain tumors and 12 (39%) without. Of the thirty-one, fifteen (48%) had NF1, 1 had Tuberous sclerosis. Cardiac toxicity consisted of asymptomatic sinus tachycardia, bradycardia, or decreased ejection fraction (EF). Thirteen patients (42%) experienced an asymptomatic decrease in left-ventricular ejection fraction (EF), Grade I-III. Time on therapy before decreased EF was 5 days to 21 months, median 2.8 months. Decreased EF developed in 5 of 13 patients receiving selumetinib and 8 of 18 receiving trametinib. Of the patients who developed decreased EF, 11 (85%) had brain tumors, 6 (46%) had NF1, and 89% had received prior systemic therapy. Out of the patients who had received no prior systemic therapy (6), 2 (33%) had decreased EF, while 11/25 (44%) of those who had received prior systemic therapy did. Drug was held temporarily for 6 patients, with dose limiting toxicity for 5 patients. Drug was discontinued for 1 patient after EF continued to decline despite dose reduction. Patients showed improvement in EF as early as 2 weeks after holding therapy. CONCLUSIONS: Cardiac toxicity in our patients was limited to asymptomatic reduction in ejection fraction, sinus bradycardia and tachycardia, reinforcing the need for appropriate monitoring via echocardiography. Prior systemic therapy was associated with decreased EF.

Phase II study of selumetinib, an orally active inhibitor of MEK1 and MEK2 kinases, in KRASG12R-mutant pancreatic ductal adenocarcinoma

SummaryBackground Preclinical evidence has suggested that a subset of pancreatic cancers with the G12R mutational isoform of the KRAS oncogene is more sensitive to MAPK pathway blockade than pancreatic tumors with other KRAS isoforms. We conducted a biomarker-driven trial of selumetinib (KOSELUGO™; ARRY-142886), an orally active, allosteric mitogen-activated protein kinase 1 and 2 (MEK1/2) inhibitor, in pancreas cancer patients with somatic KRASG12R mutations. Methods In this two-stage, phase II study (NCT03040986) patients with advanced pancreas cancer harboring somatic KRASG12R variants who had received at least one standard-of-care systemic therapy regimen received 75 mg selumetinib orally twice a day until disease progression or unacceptable toxicity occurred. The primary outcome of the study was best objective response (BOR). Results From August 2017 to February 2018 a total of 8 patients with confirmed somatic KRASG12R mutations and a median age of 61.5 years were treated with selumetinib. Seven out of eight (87.5%) had received two or more lines of prior systemic chemotherapy. After a median follow-up period of 8.5 months (range 2 to 20), three patients had stable disease for more than 6 months while receiving selumetinib. No patients achieved an objective partial response. Median progression-free survival (PFS) was 3.0 months (95% CI, 0.8–8.2) and median overall survival (OS) 9 months (95% CI, 2.5–20.9). Conclusion This study in heavily pre-treated pancreatic adenocarcinoma patients suggests alternative strategies beyond single agent MEK inhibition are required for this unique, molecular subset of pancreatic cancer patients. The trial was registered on February 2nd, 2017 under identifier NCT03040986 with ClinicalTrials.gov.

RARE-54. MEK INHIBITION FOR AGGRESSIVE GLIOMAS IN ADULTS WITH NEUROFIBROMATOSIS TYPE 1

Abstract BACKGROUND Neurofibromatosis type 1 (NF1) is caused by mutations of the tumor suppressor gene NF1 resulting in decreased levels of neurofibromin and dysregulation of the RAS pathway, including MEK/ERK upregulation. Central nervous system malignancies have an overall standardized incidence ratio of 37.5 in population studies, with higher rates of aggressive gliomas in adults with NF1. Further, the response to standard glioma therapies in this population is mixed in published series. MEK1/2 inhibitors have shown activity in both NF1 driven plexiform neurofibromas and low grade gliomas, however, data is lacking about their optimal dose or sequencing relative to standard glioma therapies for adults with NF1 associated aggressive gliomas. We present recent experiences with the MEK1/2 inhibitor trametinib in adults with NF1 and newly diagnosed aggressive gliomas. METHODS A retrospective chart review was conducted on all adult patients with NF1 and astrocytoma treated with trametinib at the Johns Hopkins Brain Cancer Program from 2016 to 2018. RESULTS Four patients met selection criteria. Median age at diagnosis was 37 years [25–43]. Histologic diagnoses were: pilocytic astrocytoma (1), diffuse astrocytomas (2), and anaplastic astrocytoma (1). All tumors were IDH1 wild-type. Three received trametinib as frontline therapy (2 mg daily), one as second line treatment. All achieved a partial radiographic response within 2 months from the start of trametinib. Median overall survival was 15 months [4–19]; three patients are alive at publication, with a median of 12 months from diagnosis. Median progression-free survival was 2.5 months [1–11]. CONCLUSIONS Astrocytomas in adults with NF1, including histologically low-grade tumors, often have a more aggressive course than suggested by histology. These cases demonstrate clinical benefit from single agent MEK inhibition as well as possible synergistic effects with conventional therapies for gliomas in people with NF1, however, prospective investigation is needed.

Trametinib for aggressive gliomas in adults with neurofibromatosis type 1.

e13562 Background: Neurofibromatosis type 1 (NF1) is caused by mutations of the tumor suppressor gene NF1 resulting in decreased levels of neurofibromin and dysregulation of the RAS pathway, including MEK/ERK upregulation. NF1 is characterized by multiple benign and malignant tumors. Central nervous system tumors have an overall standardized incidence ratio of 37.5 in population studies with high rates of aggressive gliomas in adults with NF1. The response to standard glioma therapies is mixed. MEK1/2 inhibitors have shown activity in NF1 driven plexiform neurofibromas and low grade gliomas, however, data is lacking about the optimal dose or sequencing of MEK1/2 inhibitors for adults with NF1 associated aggressive gliomas. We present recent experiences with the MEK1/2 inhibitor trametinib in adults with NF1 and aggressive gliomas. Methods: A retrospective chart review was conducted on all adult patients with NF1 and astrocytoma treated with trametinib at the Johns Hopkins Brain Cancer Program between 2016-2018. Results: Three patients met selection criteria. Two received trametinib as initial therapy (2 mg daily). All developed an acneiform rash; one had transient transaminitis requiring temporary discontinuation and subsequent dose reductions. Clinical benefit was achieved associated with two partial radiographic responses. Conclusions: Astrocytomas in adults with NF1 often have a more aggressive course than suggested by histology. The cases presented here demonstrate clinical benefit from single agent MEK inhibition as well as possible synergistic effects with conventional therapies for gliomas in this patient population. These observations highlight the need for further research in this area. [Table: see text]

Histone deacetylase inhibition in combination with MEK or BCL-2 inhibition in multiple myeloma.

Despite recent advances in the treatment of multiple myeloma, patients with this disease still inevitably relapse and become refractory to existing therapies. Mutations in K-RAS, N-RAS and B-RAF are common in multiple myeloma, affecting 50% of patients at diagnosis and >70% at relapse. However, targeting mutated RAS/RAF via MEK inhibition is merely cytostatic in myeloma and largely ineffective in the clinic. We examined mechanisms mediating this resistance and identified histone deacetylase inhibitors as potent synergistic partners. Combining the MEK inhibitor AZD6244 (selumetinib) with the pan-histone deacetylase inhibitor LBH589 (panobinostat) induced synergistic apoptosis in RAS/RAF mutated multiple myeloma cell lines. Interestingly, this synergy was dependent on the pro-apoptotic protein BIM. We determined that while single-agent MEK inhibition increased BIM levels, the protein remained sequestered by antiapoptotic BCL-2 family members. LBH589 dissociated BIM from MCL-1 and BCL-XL, which allowed it to bind BAX/BAK and thereby initiate apoptosis. The AZD6244/LBH589 combination was specifically active in cell lines with more BIM:MCL-1 complexes at baseline; resistant cell lines had more BIM:BCL-2 complexes. Those resistant cell lines were synergistically killed by combining the BH3 mimetic ABT-199 (venetoclax) with LBH589. Using more specific histone deacetylase inhibitors, i.e. MS275 (entinostat) and FK228 (romidepsin), and genetic methods, we determined that concomitant inhibition of histone deacetylases 1 and 2 was sufficient to synergize with either MEK or BCL-2 inhibition. Furthermore, these drug combinations effectively killed plasma cells from myeloma patients ex vivo. Given the preponderance of RAS/RAF mutations, and the fact that ABT-199 has demonstrated clinical efficacy in relapsed/refractory multiple myeloma, these drug combinations hold prom ise as biomarker-driven therapies.

Inhibition of MEK and DDR Pathways Induces Synergistic Killing of Novel Mll-Af4 B-ALL Model Harboring Activated Ras Mutations

Childhood B-cell acute lymphoblastic leukemia (B-ALL) that harbor a translocation of the MLL1 and AF4 genes are considered high-risk with poor prognosis (event-free survival (EFS) of 35%-50%), especially when compared to non-MLL-rearranged (MLL-R) childhood ALL (EFS >85%). An important obstacle to developing new therapeutic approaches for this patient population is the lack of models that faithfully recapitulate the short latency and aggressiveness of this disease. Recently, whole genome sequencing of patient childhood MLL-R leukemias revealed that activating mutations of the proto-oncogenes involved in signaling, most prominently, N or K-RAS were found in nearly 50% of patients. Patients with these co-occurring mutations have an even poorer overall survival rate, indicating that a model harboring both mutations is of extreme interest.Here, we report the generation of a highly aggressive, serially transplantable B-ALL by the retroviral overexpression of activating N-RasG12D mutant in bone marrow of an inducible knock-in Mll-Af4 murine model that we have previously published. Recipient mice injected with Mll-Af4/N-RasG12Dpre-leukemic bone marrow cells developed an acute B-ALL (B220+CD43+IgM-) with short latency to development of disease (median 35 days). Furthermore, the resultant primary B-ALL was serially transplantable into sub-lethally irradiated recipients with accelerated latency to secondary and tertiary disease developing at a median of 20 and 12 days, respectively.As our model includes an activating mutation in N-Ras, we wanted to see if the cells would be sensitive to small molecule inhibitors of downstream effectors of Ras. Pre-leukemic Mll-Af4/N-RasG12D cells were sensitive to two different MEK inhibitors, Trametinib or PD901, in vitro. Furthermore, in vivo treatment of tertiary B-ALL mice with Trametinib showed significant reduction in leukemia burden after 7 days of treatment, as well as increase in survival, compared to vehicle controls. However, prolonged in vivo treatment with Trametinib eventually led to loss of sensitivity and development of B-ALL in our mouse model, suggesting that Trametinib alone is insufficient to prevent leukemia progression.As single agent MEK inhibition was insufficient to generate long-term durable responses, we conducted RNA-Sequencing of primary Mll-Af4/N-RasG12D leukemias to discover pathways amenable for therapeutic intervention. Gene set enrichment analysis suggested that targeting the DNA damage response (DDR) pathway as an attractive therapeutic opportunity. We were able to demonstrate an increased basal level of replicative stress in our Mll-Af4/NrasG12D pre-leukemic cells and sensitivity to small molecule inhibition of ATR, a master regulator of the G2 to M transition of cell cycle progression, with AZ20, a selective ATR inhibitor. In vitro and in vivo treatment with AZ20 led to increased leukemia cytotoxicity. However, similar to Trametinib treatment, tertiary B-ALL mice eventually succumbed to disease with prolonged AZ20 treatment in vivo.Since neither single agent MEK nor ATR inhibition could prevent leukemic progression in vivo, we tested the combination and found increased cytotoxicity and cell cycle arrest in vitro at concentrations well below the IC50, as compared to single agent treatment. In vivocombination treatment also demonstrated decreased leukemia burden and significant prolonged survival compared to either AZ20 or Trametinib alone. Lastly, we tested out the efficacy of combination therapy in human B-ALL patient derived xenograft harboring both MLL-AF4 and activating N-RASmutations. 250,000 human primary leukemic blasts were transplanted into non-irradiated immune-compromised mice and treated with vehicle, single agent, or the combination for 14 days. Similar to the results seen in our mouse model, combination treatment with Trametinib and AZ20 led to significant reductions in leukemic burden.In summary, our model of B-ALL faithfully recapitulates the short latency and aggressiveness of this disease and was predictive of response in human patient samples harboring MLL-AF4 and activating N-RAS mutations to small molecule inhibitor therapy to MEK and DDR pathways. In the future, this model can be used as a platform to not only better understand the molecular events governing and sustaining leukemogenesis, but also as a discovery platform for novel therapeutic combinations. DisclosuresArmstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

Abstract 4905: Effects of MAPK pathway inhibitors in the tumor immune microenvironment

Abstract Immunotherapeutic agents have shown great promise in the clinic in recent years and this has led to their approval as single agents or as immune doublet combinations in melanoma and lung cancer. In order to increase the extent of benefit from these agents and to extend immunotherapies to additional patients, combinations are being evaluated of immunotherapeutic agents with chemotherapy and targeted agents. Inhibitors of the mitogen-activated kinase protein kinase (MAPK) pathway, including BRAF and MEK inhibitors, have been approved in melanoma, and are being evaluated in additional indications. We evaluated the effects of MEK and ERK inhibitors on human T cells cultured in vitro as well as on the tumor microenvironment in preclinical syngeneic mouse models. In studies with human T cells in vitro, we find that MEK or ERK inhibition reduced proliferation of naïve T cells, but had little effect on the proliferation of central memory cells, suggesting a differential requirement on MAPK signaling for proliferation of these T cell subsets. In vivo, MEK and ERK inhibition resulted in an increase in CD8+ infiltration in the tumor, as well as a decrease of markers of T cell exhaustion such as PD-1 and EOMES. The combination of MEK or ERK inhibitors plus anti-PD-L1 resulted in increased CD8+ effector function as measured by increased interferon gamma (IFNg) levels. In some cases, the MEK and ERK inhibitors showed differences in their pharmacodynamic effects as single agents; however, in combination with anti-PD-L1, the effects were similar. For example, single agent MEK inhibition but not single agent ERK inhibition increased the number of CD4+ helper and regulatory T cells (Tregs) in the tumor. However, both of these CD4+ subsets, as well as CD4+ IFNg levels, were increased with MEK or ERK inhibitors combined with anti-PD-L1. Similarly, single agent MEK inhibition decreased the number of infiltrating CD11b+Ly6G+ myeloid cells whereas single agent ERK inhibition increased the number of infiltrating CD11b+Ly6C+ myeloid cells. However, in combination with anti-PD-L1, these changes in myeloid populations were less apparent. Taken together, these data show that MAPK pathway suppression can modulate multiple immune cell subtypes within the tumor, with many of these changes expected to activate the immune infiltrate. Despite the increase in Tregs in response to ERK and MEK inhibition in combination with anti-PD-L1, these combinations were efficacious. This suggests that the other immune stimulatory effects of these treatments are sufficient to drive efficacy despite the presence of increased Tregs. Our data suggest that the combination of MAPK inhibition plus anti-PD-L1 inhibition, such as with atezolizumab, is a promising hypothesis to test in the clinic. Citation Format: Marcia P. Belvin, Erin Williams, Shiuh-Ming Luoh, Jeanne Cheung, Christine Orr, Emily Chan, Peter Ebert, Ira Mellman, Jeong Kim, Mark Merchant. Effects of MAPK pathway inhibitors in the tumor immune microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4905.

Abstract B42: Targeting PI3K in personalized treatment of BRAF-mutated pediatric low-grade gliomas

Abstract Introduction: Pediatric low-grade gliomas (PLGG) constitute the most common group of central nervous system tumors in children. Although they exhibit rare malignant transformation and relatively slow growth, PLGGs pose great morbidity. Viewed as a chronic disease, ideal therapy for PLGG should carry limited side effects that can be best achieved through selective, personalized therapy. Despite the known heterogeneity of PLGGs and characterized driver mutations that together offer the perfect, timely platform for personalized approaches to therapy, most children are still treated with standard chemotherapy protocols in a “one treatment fits all” approach. Recent genomic studies have defined recurrent, diverse alterations driving WHO grade 1 and 2 PLGGs. Activation of mitogen protein kinase (MAPK) and phosphatidylinositol-3′ kinase (PI3K) in PLGGs suggests the potential efficacy of agents that target these key growth regulatory pathways. BRAF, a kinase within the MAPK pathway, is activated by missense mutation (V600E) in ∼20% of grade 2-4 pediatric gliomas or KIAA1549-BRAF fusion in ∼60% of pilocytic astrocytomas. In addition to RAS/MEK/MAPK/ERK, the PI3K/AKT/mTOR signaling cascade is activated in ∼half of PLGGs. We hypothesize that knowledge of individual tumor BRAF genotype can guide selection of PI3K/MAPK inhibitors as single agents and in combinations, to maximize efficacy of therapy and overcome innate and acquired resistance to targeted agents. Methods: We used human glioma cell lines containing BRAFV600E (AM38, DBT-RG, BT40), or wild-type BRAF (BRAFWT; SF188, SF9427, SF9402) and isogenic systems of KIAA1549:BRAF-expressing NIH3T3 cells and BRAFV600E-expressing murine brain tumors. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK). Cell cycle distribution and apoptosis were assessed using flow cytometry; proliferation was determined using an ATP-based assay (CellTiter-Glo). In vivo activity of these inhibitors was assessed in the BT40 PLGG xenograft mouse model. Results: In BRAFV600E cells, combination of PLX4720+everolimus and PLX4720+AZD6244 exhibited significantly greater effects on cell viability, apoptosis, and cell cycle than respective single agents. In BRAFWT cells, everolimus+AZD6244 was superior compared to respective monotherapies. Similar results were found using isogenic murine cells. In KIAA1549:BRAF fusion cells, MEK inhibition reduced cell viability and S-phase content, effects that were modestly augmented by mTOR inhibition. In vivo experiments in the PLGG xenograft model BT40 showed the greatest survival advantage in mice treated with combination of AZD6244+PLX4720 or AZD6244+everolimus compared with respective monotherapies (p<0.01). Conclusions: For BRAFV600E tumors, combination of everolimus+MEK or PLX4720+MEK inhibitors is equally efficacious and superior to single agents, and the choice between these two might be dictated by clinical tolerability. For BRAFWT PLGGs, combination of everolimus+MEK inhibitor is superior to single agent therapies. Optimal treatment of KIAA1549:BRAF-expressing PLGGs is still under investigation as single agent MEK inhibition is extremely efficacious but mTOR inhibition may contribute to greater overall anti-neoplastic effects. Citation Format: Aleksandra K. Olow, Sabine Mueller, Xiaodong Yang, Rintaro Hashizume, William Weiss, Adam C. Resnick, Angela J. Sievert, Mitchel S. Berger, Nalin Gupta, David C. James, Daphne A. Haas-Kogan. Targeting PI3K in personalized treatment of BRAF-mutated pediatric low-grade gliomas. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B42.

Abstract A28: Apoptosis induction with the MEK inhibitor AZD6244 and BH3 mimetic ABT-737 in melanoma.

Abstract Over sixty percent of patients with melanoma have the activating B-RAFV600E mutation leading to increased activation of the mitogen activated protein kinase (MAPK) pathway. The downstream effects of MAPK pathway activation are increased proliferation, invasiveness, and survival through the regulation of down-stream targets such as the pro-apoptotic protein BIM. Prior studies have shown that inhibiting MAPK kinase (MEK), downstream of constitutively active B-RAF, can up-regulate BIM, leading to increased apoptosis in melanoma cell lines. However, single agent MEK inhibition has had limited effectiveness in the clinical setting for late-stage melanoma patients, likely due to an inadequate induction of apoptosis. We hypothesized that combining the clinically relevant MEK inhibitor, AZD6244 (ARRY142886) with a BCL-2 inhibitor, ABT-737, would lead to increased apoptosis over single agent therapies in melanoma cells via BIM activation and pan-BCL-2 inhibition. We compared single treatments of AZD6244 and ABT737 to the combination of these agents in a panel of melanoma tumor cell lines chosen by mutational status in both a monolayer system and an anchorage independent system. The combination of AZD6244 and ABT-737 had a synergistic effect on decreasing cell viability and increasing apoptosis compared to single agent treatments regardless of B-RAF mutational status. Western blotting revealed dramatic increases in cleaved caspase 3 and PARP with combinational treatment compared to single agent therapy. These early pre-clinical experiments indicate that AZD6244 combined with ABT-737 may be an effective combination therapy for patients with late-stage melanoma. Currently, we have plans to conduct in vivo studies to translate these results into the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A28.