NRAS-mutant Melanoma Research Articles

Abstract 3099: A new HTS ELISA for monitoring NRAS activity to expedite drug discovery

Abstract Oncogenic mutations in the KRAS, HRAS, and NRAS isoforms of the RAS gene family alter GTP binding and hydrolysis leading to errant over activation of the MAPK/ERK pathway. While the total of all such RAS mutations drives some 20-25% of all human cancers, each isoform tends to favor particular mutations that are cancer specific. Many evolving treatment strategies for these cancers target these mutant isoforms to minimize off target effects. NRAS mutations are seen in about one-fifth of cutaneous melanomas and patients with NRAS mutation melanomas have a poorer prognosis due to the high aggressiveness of RAS mutant tumors, a lack of efficient targeted therapies, or rapidly emerging resistance to existing treatments. Furthermore, there is no currently authorized targeted treatment for NRAS-mutated melanoma. As a result, there is a growing need for assays which can quantify activated NRAS. Here we describe the development of a highly specific and sensitive ELISA for determining the active NRAS protein in high throughput compatible (HTS) format. A NRAS specific recombinant antibody was developed via cloning of immunoglobulin variable regions from antigen positive B cells isolated by FACS from rabbits immunized with a NRAS peptide corresponding to a region of low homology between the RAS isoforms. Immunoblotting and a bead-based multiplex ELISA with all three RAS isoforms were used to confirm antibody specificity with recombinant HRAS, KRAS, and NRAS proteins over a wide concentration range. The new immunoassay enables in-well lysis of cells cultivated in 96-well plate. Activated NRAS in the lysate is collected on the test plate and detected using the NRAS specific antibody via chemiluminescence. We anticipate that this assay will further the discovery of novel NRAS specific therapeutics. EGF treatment of MCF7 cells demonstrated activation in cells expressing wildtype NRAS in preliminary assay validation. Evaluation of assay performance in mutant NRAS melanoma cell lines will be presented. Citation Format: Hyun Min Park, Julie Weber, Michelle Carlson, Mary Anne Jelinek. A new HTS ELISA for monitoring NRAS activity to expedite drug discovery [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 3099.

A phase II study of efficacy and safety of the MEK inhibitor tunlametinib in patients with advanced NRAS-mutant melanoma

BackgroundNRAS-mutant melanoma is an aggressive subtype with poor prognosis; however, there is no approved targeted therapy to date worldwide. MethodsWe conducted a multicenter, single-arm, phase II, pivotal registrational study that evaluated the efficacy and safety of the MEK inhibitor tunlametinib in patients with unresectable, stage III/IV, NRAS-mutant melanoma (NCT05217303). The primary endpoint was objective response rate (ORR) assessed by independent radiological review committee (IRRC) per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. The secondary endpoints included progression-free survival (PFS), disease control rate (DCR), duration of response(DOR), overall survival (OS) and safety. FindingsBetween November 2, 2020 and February 11, 2022, a total of 100 patients were enrolled. All (n = 100) patients received at least one dose of tunlametinib (safety analysis set [SAS]) and 95 had central laboratory-confirmed NRAS mutations (full analysis set [FAS]). In the FAS, NRAS mutations were observed at Q61 (78.9%), G12 (15.8%) and G13 (5.3%). The IRRC-assessed ORR was 35.8%, with a median DOR of 6.1 months. The median PFS was 4.2 months, DCR was 72.6% and median OS was 13.7 months. Subgroup analysis showed that in patients who had previously received immunotherapy, the ORR was 40.6%. No treatment-related deaths occurred. InterpretationTunlametinib showed promising antitumor activity with a manageable safety profile in patients with advanced NRAS-mutant melanoma, including those who had prior exposure to immunotherapy. The findings warrant further validation in a randomized clinical trial.

The therapeutically actionable long non-coding RNA ‘T-RECS’ is essential to cancer cells’ survival in NRAS/MAPK-driven melanoma

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impact on normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

NRAS Mutant Dictates AHCYL1-Governed ER Calcium Homeostasis for Melanoma Tumor Growth.

Our findings suggest that targeting the AHCYL1-IP3R axis presents a novel therapeutic approach for NRAS-mutated melanomas, with potential applicability to all cancers harboring RAS mutations, such as KRAS-mutated human colorectal cancers.

Augmenting MEK inhibitor efficacy in BRAF wild-type melanoma: synergistic effects of disulfiram combination therapy

BackgroundMEK inhibitors (MEKi) were shown to be clinically insufficiently effective in patients suffering from BRAF wild-type (BRAF WT) melanoma, even if the MAPK pathway was constitutively activated due to mutations in NRAS or NF-1. Thus, novel combinations are needed to increase the efficacy and duration of response to MEKi in BRAF WT melanoma. Disulfiram and its metabolite diethyldithiocarbamate are known to have antitumor effects related to cellular stress, and induction of endoplasmic reticulum (ER) stress was found to synergize with MEK inhibitors in NRAS-mutated melanoma cells. Therefore, we investigated the combination of both therapeutics to test their effects on BRAF-WT melanoma cells and compared them with monotherapy using the MEKi trametinib.MethodsThe effects of combined therapy with disulfiram or its metabolite diethyldithiocarbamate and the MEKi trametinib were evaluated in a series of BRAF-WT melanoma cell lines by measuring cell viability and apoptosis induction. Cytotoxicity was additionally assessed in 3D spheroids, ex vivo melanoma slice cultures, and in vivo xenograft mouse models. The response of melanoma cells to treatment was studied at the RNA and protein levels to decipher the mode of action. Intracellular and intratumoral copper measurements were performed to investigate the role of copper ions in the antitumor cytotoxicity of disulfiram and its combination with the MEKi.ResultsDiethyldithiocarbamate enhanced trametinib-induced cytotoxicity and apoptosis induction in 2D and 3D melanoma culture models. Mechanistically, copper-dependent induction of oxidative stress and ER stress led to Janus kinase (JNK)-mediated apoptosis in melanoma cells. This mechanism was also detectable in patient-derived xenograft melanoma models and resulted in a significantly improved therapeutic effect compared to monotherapy with the MEKi trametinib.ConclusionsDisulfiram and its metabolite represent an attractive pharmaceutical approach to induce ER stress in melanoma cells that potentiates the antitumor effect of MEK inhibition and may be an interesting candidate for combination therapy of BRAF WT melanoma.

Targeted Combination Helps Patients With NRAS-Mutated Melanoma

Targeted Combination Helps Patients With NRAS-Mutated Melanoma

First-in-Human Dose-Escalation Study of the Novel Oral Depsipeptide Class I-Targeting HDAC Inhibitor Bocodepsin (OKI-179) in Patients with Advanced Solid Tumors.

(1) Background: Histone deacetylases (HDACs) play a critical role in epigenetic signaling in cancer; however, available HDAC inhibitors have limited therapeutic windows and suboptimal pharmacokinetics (PK). This first-in-human phase I dose escalation study evaluated the safety, PK, pharmacodynamics (PDx), and efficacy of the oral Class I-targeting HDAC inhibitor bocodepsin (OKI-179). (2) Patients and Methods: Patients (n = 34) with advanced solid tumors were treated with OKI-179 orally once daily in three schedules: 4 days on 3 days off (4:3), 5 days on 2 days off (5:2), or continuous in 21-day cycles until disease progression or unacceptable toxicity. Single-patient escalation cohorts followed a standard 3 + 3 design. (3) Results: The mean duration of treatment was 81.2 (range 11-447) days. The most frequent adverse events in all patients were nausea (70.6%), fatigue (47.1%), and thrombocytopenia (41.2%). The maximum tolerated dose (MTD) of OKI-179 was 450 mg with 4:3 and 200 mg with continuous dosing. Dose-limiting toxicities included decreased platelet count and nausea. Prolonged disease control was observed, including two patients with platinum-resistant ovarian cancer. Systemic exposure to the active metabolite exceeded the preclinical efficacy threshold at doses lower than the MTD and was temporally associated with increased histone acetylation in circulating T cells. (4) Conclusions: OKI-179 has a manageable safety profile at the recommended phase 2 dose (RP2D) of 300 mg daily on a 4:3 schedule with prophylactic oral antiemetics. OKI-179 is currently being investigated with the MEK inhibitor binimetinib in patients with NRAS-mutated melanoma in the phase 2 Nautilus trial.

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

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

Abstract PR012: Novel strategy for RAS-pathway targeting: Initial results from a phase 1b/2 clinical trial of the oral HDAC inhibitor bocodepsin (OKI-179) combined with binimetinib in patients with RAS-pathway mutated solid tumors and NRAS-mutated melanoma

Abstract Background: Activating RAS pathway mutations occur in > 30% of human cancers, including NRAS mutations in 15-20% of advanced melanoma. MEK inhibitors have modest single agent activity in NRAS-mutated melanoma, showing a 15% overall response rate and 2.8 month progression free survival. Bocodepsin (OKI-179) is a novel Class I-selective, oral histone deacetylase inhibitor (HDACi). The combination of bocodepsin and binimetinib is preferentially synergistic in RAS pathway mutated tumor models, leading to the unique mechanism of double strand DNA breaks and cellular apoptosis. Here we present the initial results of the Nautilus phase 1b/2 trial of bocodepsin and binimetinib (NCT05340621). Materials and Methods: In Phase 1b, patients with previously treated advanced solid tumors with any activating RAS pathway mutation were treated with binimetinib oral 45 mg twice daily with bocodepsin 200 mg (cohort 1) or 300 mg (cohort 2) oral daily 4 days on 3 days off in 28-day cycles. In Phase 2, patients with NRAS-mutated melanoma previously treated with immunotherapy were treated with bocodepsin 300 mg with binimetinib in a Simon optimal 2-stage study with a primary endpoint of objective response rate. Secondary endpoints included safety and PK. Results: As of July 6, 2023, 14 patients were enrolled in Phase 1b in 2 cohorts and across multiple tumor types (colorectal, n=4; melanoma, n=3; NSCLC, n=2; pancreatic, n=2; other, n=3) and RAS-pathway mutations (KRAS, n=9; NRAS, n=3; BRAF, n=1; NF1, n=1). The median age was 70 (41-76) and median number of prior therapies was 6 (1-13). No DLTs were observed. Bocodepsin 300 mg was identified as the RP2D in combination with standard dose binimetinib. The most common treatment-emergent adverse events were consistent with either binimetinib or bocodepsin alone known adverse events and included diarrhea (N=11, 78.57%), nausea (N=9, 64.29%), anemia (N=8, 57.14%), and dermatitis acneiform (N=8, 57.14%). The most common grade 3/4 events included anemia (N=4, 28.57%) and syncope (N=2, 14.29%). Of the 14 evaluable patients in Phase 1, the best response was partial response (PR) in 1 pt (NRAS-mutated melanoma) and stable disease (SD) in 6 pts, with a median time on study of 120 days (50-260). Phase 2 enrollment is ongoing. At the data cutoff, in the 9 response evaluable NRAS melanoma patients (between Ph1 and Ph2), 3 PR and 3 SD were observed. Conclusions: The combination of bocodepsin and binimetinib in patients with RAS-mutated advanced cancers is tolerable with manageable AEs. Initial response data in NRAS melanoma are supportive of potential combinatorial activity of binimetinib and bocodepsin. The data for Phase 2 will be updated at the time of presentation. Citation Format: Rodabe N Amaria, Herbert Duvivier, Katy Tsai, Robert Galamaga, Parisa Momtaz, Evan Pisick, Natalie Langr, Harish Dave, Duncan Walker, Jennifer Diamond, Kevin Litwiler, Ryan Sullivan. Novel strategy for RAS-pathway targeting: Initial results from a phase 1b/2 clinical trial of the oral HDAC inhibitor bocodepsin (OKI-179) combined with binimetinib in patients with RAS-pathway mutated solid tumors and NRAS-mutated melanoma [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 PR012.

Abstract PR009: Discovery of covalent NRAS inhibitors targeting the Palmitoylation site through imaging-based high-throughput screening

Abstract NRAS mutations play a crucial role as oncogenic drivers of tumor cell growth and chemo-resistance in melanoma and acute myeloid leukemia (AML). In melanoma, NRAS mutations occur in approximately 25% of cases, and are generally mutually exclusive with BRAF-mutations. NRAS mutant tumors often exhibit an aggressive phenotype, such as thicker lesions, increased mitotic activity, and higher rates of lymph node metastasis. Surgical resection of NRAS mutant melanoma has a poor prognosis with a lower median overall survival (OS) compared to non-NRAS-mutant melanoma (8.2 vs. 15.1 months for stage IV disease). Current treatment strategies for stage III/IV non-resectable metastatic NRAS mutant melanoma include immunotherapy and indirect targeting of NRAS associated signaling pathways, highlighting the urgent need to develop small molecule NRAS inhibitors. NRAS palmitoylation is essential for its oncogenic function by regulating its trafficking and anchoring to the plasma membrane. Unlike other RAS isoforms, NRAS relies on a single palmitoylation site (C181) for its plasma membrane localization and this unique dependency makes NRAS more sensitive to palmitoylation inhibitors. To identify selective NRAS inhibitors that covalently modify C181 and prevent its palmitoylation, we employed an imaging-based NRAS translocation high-throughput screen using a library of 23,000 covalent molecules. We identified a set of covalent inhibitors that selectively target NRAS translocation but do not affect HRAS localization. We further confirmed the direct binding of these compound hits to NRAS at its palmitoylation site (C181) using intact mass spectrometry and chemo-proteomic peptide mapping techniques. Moreover, we validated the functional impact of these compound hits by demonstrating their selective inhibition of cell growth in mutant NRAS-driven melanoma cell lines. In conclusion, our study presents the first direct inhibitor for NRAS, discovered through a high-throughput covalent screening approach. This finding holds promising implications for the development of novel targeted therapies for NRAS mutant tumors. Citation Format: Zhao Wang, Keith Moore, Simon Ng, Chao Wang, Ronald Tomlinson, Huay-Keng Loke, Jamie Rice, Ivan Cornella. Discovery of covalent NRAS inhibitors targeting the Palmitoylation site through imaging-based high-throughput screening [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 PR009.

Abstract A091: The class I selective, oral HDAC inhibitor bocodepsin enhances the response to MAPK pathway inhibitors in multiple tumor types with mutations in MAPK pathway signaling proteins

Abstract Activating MAPK pathway mutations, found in >30% of all human tumors, are frequently associated with poor outcomes. Several MAPK pathway targeted drugs are approved but single agent activity is modest, supporting the need for rational targeted combinations to improve outcomes. Multiple studies propose synthetic lethality between Class I HDAC inhibitors and MAPK pathway inhibitors in MAPK pathway mutated cancers, due to a combined effect of inhibiting double-stranded DNA repair and other survival pathways to drive apoptosis. The use of HDACi has been challenged by poor potency, selectivity, safety and convenience, highlighting the need for a better HDACi in the clinic. Bocodepsin, a potent class I-selective oral HDACi, is currently in a phase 1b/2 trial in combination with binimetinib in patients with NRAS mutant melanoma. The clinical profile shows exposure consistent with preclinical activity with strong pharmacodynamic activity at tolerated doses. OKI-005, a bocodepsin analog optimized for in vitro activity, produces the same active metabolite as bocodepsin. To explore the potential for broad application of bocodepsin combinations, we evaluated OKI-005 in combination with the MEK inhibitor binimetinib in a 72H in vitro proliferation screen of 89 cell lines covering a range of tumor types, MAPK pathway mutations, and co-mutations. Net cell killing with the combination vs. single agents was evaluated at clinically relevant doses. Combination activity was observed in tumor types that commonly harbor activating MAPK pathway mutations, including melanoma (6/13, 46%), pancreatic (3/5, 60%), lung (10/22, 45%) and colon (8/13, 62%). Among these, single agent binimetinib activity was observed in only 1/53. The combination response rate in colon cancer models was of note, as single agent MAPK pathway inhibitors are typically inactive in colon cancers. Activity was independent of the activating MAPK pathway mutation: combination activity was observed in lines with KRAS (13/24, 52%), NRAS (8/12, 67%) and BRAF (7/13, 54%) mutations. These findings translated to combinations with other MAPK inhibitors in vitro and in vivo. In vitro, OKI-005 exhibited enhanced activity in combination with KRAS inhibitors and with the ERK inhibitor GDC-0994. 25 of the 89 tumor lines were evaluated in combination with GDC-0994, and the profile of inhibition was identical to that with binimetinib. In vivo, the combination of binimetinib + bocodepsin led to 9/10 regressions in the NRAS mutant melanoma model SK-MEL-2, vs. 0/10 and 2/10 with binimetininb and bocodepsin, respectively. The combination of sotorasib + bocodepsin led to deeper regressions, vs. single agent sotorasib, in the KRAS mutant NSCLC model H358, and greater response in the KRAS mutant NSCLC model H2122. Combination activity was associated with increases in cleaved PARP and p-gH2AX, markers of DNA damage induction and apoptosis. These data demonstrate the potential for bocodepsin to synergize with MAPK pathway inhibitors across multiple indications in tumors that harbor MAPK pathway mutations. Citation Format: Rich Woessner, Maria Hoh, James Winkler, Tony Piscopio, Duncan Walker. The class I selective, oral HDAC inhibitor bocodepsin enhances the response to MAPK pathway inhibitors in multiple tumor types with mutations in MAPK pathway signaling proteins [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 A091.

Abstract B155: IK-595, a best-in-class MEK-RAF complex inhibitor, drives broad and potent anti-tumor activity in RAS/RAF-driven tumors

Abstract Alterations in the RAS/RAF/MEK/ERK pathway are the most common drivers of oncogenesis. Although MEK is a clinically validated cancer target and several MEK inhibitors have been approved by the FDA, their clinical utility has been limited to BRAF V600 mutant cancers and NF1 mutant neurofibromas. We developed IK-595, a potent inhibitor of the MEK-RAF complex, to overcome the limitations of available MEK inhibitors. IK-595 traps MEK in an inactive complex with all RAF isoforms and blocks RAF-dependent MEK phosphorylation, thereby alleviating CRAF-mediated MEK reactivation that hinders the efficacy of approved MEK inhibitors in RAS/RAF-driven tumors. We demonstrate that IK-595 leads to potent and prolonged inhibition of MEK and ERK1/2 phosphorylation, in KRAS-mutant cancer and NRAS-mutant melanoma models. In addition to trapping all three RAF isoforms, IK-595 stabilizes the interaction of MEK with BRAF Class I, II, and III mutant proteins. This delays the onset of known resistance mechanisms to other MAPK pathway targeting agents, such as CRAF-mediated pathway reactivation and receptor tyrosine kinase upregulation and/or activation. Importantly, IK-595 has a much slower off-rate binding to MEK and retains CRAF longer in an inactive complex with MEK than other MEK/RAF inhibitors allowing for more prolonged target engagement and durable pathway inhibition in RAS/RAF mutant cancer cells. Moreover, CRAF-ARAF heterodimerization, which is detected in type II pan-RAF inhibitor-treated KRAS mutant cells as an established resistance mechanism, is not observed upon IK-595 treatment. IK-595 exhibits potent signal agent activity across a wide range of cancer models harboring various RAS/RAF alterations, including lung, pancreatic, colorectal cancer (CRC), melanoma, AML, and others. Strong synergy with inhibitors targeting multiple key mechanisms, both within the RAS/MAPK pathway and across parallel survival pathways, has also been observed in multiple models. Key to the design of IK-595 is its PK profile that enables transient high plasma drug exposure and flexible dosing schedules. Intermittent dosing of IK-595 every other day (QOD) or every three days (Q3D) in KRAS mutant mouse tumor models shows similar efficacy and improved tolerability compared to daily dosing (QD), affording a larger therapeutic window. Preclinical pharmacology studies provide clear guidance to the clinical development plan of IK-595, where flexible dosing schedules and multiple expansion cohorts in RAS/RAF altered patient populations will be explored. IK-595 is a novel MEK-RAF complex inhibitor that prolongs pathway inhibition, minimizing the potential for resistance, while providing an optimal therapeutic window for patients with RAS/RAF-driven cancer. Citation Format: Eric Haines, Rachel Catterall, Victor De Jesus, Bin Li, Sarah R Wessel, Jill Cavanaugh, Michael Burke, Joseph D Manna, Okzana Zavidij, Ao Yang, Lan Xu, Sergio Santillana, Jeffrey Ecsedy, X. Michelle Zhang, Sabine K Ruppel. IK-595, a best-in-class MEK-RAF complex inhibitor, drives broad and potent anti-tumor activity in RAS/RAF-driven tumors [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 B155.

Exploring the In Vitro and In Vivo Therapeutic Potential of BRAF and MEK Inhibitor Combination in NRAS-Mutated Melanoma.

Patients with NRAS-mutant metastatic melanoma often have an aggressive disease requiring a fast-acting, effective therapy. The MEK inhibitor binimetinib shows an overall response rate of 15% in patients with NRAS-mutant melanoma, providing a backbone for combination strategies. Our previous studies demonstrated that in NRAS-mutant melanoma, the antitumor activity of the MEK inhibitor binimetinib was significantly potentiated by the BRAFV600E/K inhibitor encorafenib through the induction of ER stress, leading to melanoma cell death by apoptotic mechanisms. Encorafenib combined with binimetinib was well tolerated in a phase III trial showing potent antitumor activity in BRAF-mutant melanoma, making a rapid evaluation in NRAS-mutant melanoma imminently feasible. These data provide a mechanistic rationale for the evaluation of binimetinib combined with encorafenib in preclinical and clinical studies on NRAS-mutant metastatic melanoma. The combination of BRAFi plus MEKi was tested in a monolayer culture of patient-derived cell lines and in corresponding patient-derived tissue slice cultures of NRAS-mutant melanoma. To investigate the treatment in vivo, NSG (NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were subcutaneously injected with three different BRAF wild-type melanoma models harboring oncogenic NRAS mutations and treated orally with encorafenib (6 mg/kg body weight, daily) with or without binimetinib (8 mg/kg body weight, twice daily). In parallel, an individual healing attempt was carried out by treating one patient with an NRAS-mutated tumor. Encorafenib was able to enhance the inhibitory effect on cell growth of binimetinib only in the cell line SKMel147 in vitro. It failed to enhance the apoptotic effect found in two other NRAS-mutated cell lines. Encorafenib led to a hyperactivation of ERK which could be reduced with the combinational treatment. In two of the three patient-derived tissue slice culture models of NRAS-mutant melanomas, a slight tendency of a combinatorial effect was seen which was not significant. Encorafenib showed a slight induction of the ER stress genes ATF4, CHOP, and NUPR1. The combinational treatment was able to enhance this effect, but not significantly. In the mouse model, the combination therapy of encorafenib with binimetinib resulted in reduced tumor growth compared to the control and encorafenib groups; however, the best effect in terms of tumor growth inhibition was measured in the binimetinib therapy group. The therapy showed no effect in an individual healing attempt for a patient suffering from metastatic, therapy-refractory NRAS-mutated melanoma. In in vitro and ex vivo settings, the combination therapy was observed to elicit a response; however, it did not amplify the efficacy observed with binimetinib alone, whereas in a patient, the combinational treatment remained ineffective. The preclinical in vivo data showed no increased combinatorial effect. However, the in vivo effect of binimetinib as monotherapy was unexpectedly high in the tested regimen. Nevertheless, binimetinib proved to be advantageous in the treatment of melanoma in vivo and led to high rates of apoptosis in vitro; hence, it still seems to be a good base for combination with other substances in the treatment of patients with NRAS-mutant melanoma.

First-in-human study of naporafenib (LXH254) with or without spartalizumab in adult patients with advanced solid tumors harboring MAPK signaling pathway alterations

BackgroundWe investigated naporafenib (LXH254), a pan-RAF kinase inhibitor, with or without spartalizumab, in patients with advanced solid tumors harboring MAPK pathway alterations. MethodsThis first-in-human phase 1 study had two dose-escalation arms: single-agent naporafenib (starting at 100 mg once-daily [QD]) and naporafenib (starting at the recommended dose/regimen)/spartalizumab (400 mg every 4 weeks). The naporafenib/spartalizumab dose-expansion part enrolled patients with KRAS-mutated non-small cell lung cancer (NSCLC) and NRAS-mutated melanoma. The primary objectives were to establish the maximum tolerated doses (MTD)/recommended doses for expansion (RDE) and evaluate tolerability and safety. ResultsA total of 142 patients were included in the naporafenib dose-escalation (n = 87), naporafenib/spartalizumab dose-escalation (n = 12) and naporafenib/spartalizumab dose-expansion (n = 43) arms. The MTD/RDE of naporafenib was 600 mg twice-daily (BID). In naporafenib escalation, five patients experienced 7 dose-limiting toxicities: decreased platelet count (1200 mg QD); neuralgia, maculopapular rash, pruritus (600 mg BID); increased blood bilirubin, hyponatremia, peripheral sensory neuropathy (800 mg BID). No DLTs occurred in the naporafenib/spartalizumab arm: the RDE was established at 400 mg BID. The most common treatment-related adverse events were rash and dermatitis acneiform (each 24.1%; naporafenib), nausea and pruritus (each 33.3%; naporafenib/spartalizumab; escalation) and rash (39.5%; naporafenib/spartalizumab; expansion). Naporafenib reduced DUSP6 expression in tumors. Two partial responses (PRs) occurred in naporafenib escalation, and 1 complete response and 3 PRs in the naporafenib/spartalizumab NRAS-mutated melanoma and KRAS-mutated NSCLC arms, respectively. ConclusionsNaporafenib, with or without spartalizumab, showed an acceptable safety profile, pharmacodynamic activity and limited antitumor activity. Additional naporafenib combination therapies are currently under investigation.

A multi-omics integrative approach unravels novel genes and pathways associated with senescence escape after targeted therapy in NRAS mutant melanoma

Therapy Induced Senescence (TIS) leads to sustained growth arrest of cancer cells. The associated cytostasis has been shown to be reversible and cells escaping senescence further enhance the aggressiveness of cancers. Chemicals specifically targeting senescent cells, so-called senolytics, constitute a promising avenue for improved cancer treatment in combination with targeted therapies. Understanding how cancer cells evade senescence is needed to optimise the clinical benefits of this therapeutic approach. Here we characterised the response of three different NRAS mutant melanoma cell lines to a combination of CDK4/6 and MEK inhibitors over 33 days. Transcriptomic data show that all cell lines trigger a senescence programme coupled with strong induction of interferons. Kinome profiling revealed the activation of Receptor Tyrosine Kinases (RTKs) and enriched downstream signaling of neurotrophin, ErbB and insulin pathways. Characterisation of the miRNA interactome associates miR-211-5p with resistant phenotypes. Finally, iCell-based integration of bulk and single-cell RNA-seq data identifies biological processes perturbed during senescence and predicts 90 new genes involved in its escape. Overall, our data associate insulin signaling with persistence of a senescent phenotype and suggest a new role for interferon gamma in senescence escape through the induction of EMT and the activation of ERK5 signaling.

Single-cell transcriptomics of NRAS-mutated melanoma transitioning to drug resistance reveals P2RX7 as an indicator of early drug response

Treatment options for patients with NRAS-mutant melanoma are limited and lack an efficient targeted drug combination that significantly increases overall and progression-free survival. In addition, targeted therapy success is hampered by the inevitable emergence of drug resistance. A thorough understanding of the molecular processes driving cancer cells' escape mechanisms is crucial to tailor more efficient follow-up therapies. We performed single-cell RNA sequencing of NRAS-mutant melanoma treated with MEK1/2 plus CDK4/6 inhibitors to decipher transcriptional transitions during the development of drug resistance. Cell lines resuming full proliferation (FACs [fast-adapting cells]) and cells that became senescent (SACs [slow-adapting cells]) over prolonged treatment were identified. The early drug response was characterized by transitional states involving increased ion signaling, driven by upregulation of the ATP-gated ion channel P2RX7. P2RX7 activation was associated with improved therapy responses and, in combination with targeted drugs, could contribute to the delayed onset of acquired resistance in NRAS-mutant melanoma.

The OSUMMER lines: A series of ultraviolet-accelerated NRAS-mutant mouse melanoma cell lines syngeneic to C57BL/6.

An increasing number of cancer subtypes are treated with front-line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Preclinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of 13 C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University-Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte-specific, and clinically relevant Nras driver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non-burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune-based therapies.

Efficacy and safety of tunlametinib in patients with advanced NRAS-mutant melanoma: A multicenter, open-label, single-arm, phase 2 study.

9510 Background: NRAS-mutant melanoma is an aggressive subtype with worse prognosis. However, no targeted therapy has been approved to date worldwide. Tunlametinib (HL-085), a novel, potent, selective, oral small-molecule MEK1/2 inhibitor, has showed a favorable pharmacokinetics profile, acceptable tolerability and encouraging antitumor activity in the phase 1 study (BMC Med. 2023 Jan 4;21(1):2). Methods: This is an ongoing, multicenter, open-label, single-arm, phase 2 pivotal registration study. Patients (pts) with NRAS-mutant unresectable stage III or IV melanoma were enrolled and received tunlametinib 12 mg orally twice daily. A historical control of objective response rate (ORR) of 10% was predefined for sample size estimation (100 pts, assuming loss to follow-up rate of 5%) and efficacy evaluation. The primary endpoint was the confirmed ORR per RECIST v1.1 assessed by independent radiology review committee. Results: A total of 100 pts were enrolled. All pts were included in the safety analysis set and 95 pts were included in the full analysis set (FAS) for efficacy analysis. At cut-off date (August 17, 2022), median follow-up was 7.9 months (95% CI: 6.6, 9.8). In the FAS, the median age was 58.0 years (range: 24 to 84). Sixty-four pts (67.4%) had received prior immunotherapy (PD-1/PD-L1 inhibitor). Fifty-six pts (58.9%) were acral melanoma, 16 (16.8%) mucosal melanoma and 12 (12.6%) skin melanoma. Fourteen pts (14.7%) were stage III and 81 (85.3%) stage IV. The most frequent NRAS mutation types were Q61R (40.0%), Q61K (29.5%) and G12D (9.5%). Confirmed ORR was 34.7% (95%CI: 25.3%, 45.2%). Median progression-free survival was 4.2 months (95%CI: 3.5, 5.6), overall survival was immature, and 1-year survival rates was 57.2% (95% CI, 44.7%, 67.8%). Subgroup analysis showed that, in pts who had previously received immunotherapy, the confirmed ORR was 39.1% (95% CI: 27.1%, 52.1%). The most frequent treatment related adverse events (TRAEs) were increased blood creatine phosphokinase (CK), diarrhea, peripheral oedema, facial oedema and increased aspartate aminotransferase. Grade ≥3 TRAEs occurred in 68 pts (68.0%), of which 38.0% (38/100) were increased blood CK. Most of pts with CK elevation are asymptomatic and can be managed by dose interruption or reduction without permanent treatment discontinuation. No treatment-related death was reported. Conclusions: Tunlametinib was well tolerated and demonstrated encouraging treatment response rate in pts with advanced NRAS-mutant melanoma. These results indicate that tunlametinib could be a promising treatment option for NRAS-mutant melanoma, even for those immunotherapy failed pts. Clinical trial information: NCT05217303 .

Deconstructing the role of MALAT1 in MAPK-signaling in melanoma: insights from antisense oligonucleotide treatment.

The long non-coding RNA (lncRNA) MALAT1 is a regulator of oncogenesis and cancer progression. MAPK-pathway upregulation is the main event in the development and progression of human cancer, including melanoma and recent studies have shown that MALAT1 has a significant impact on the regulation of gene and protein expression in the MAPK pathway. However, the role of MALAT1 in regulation of gene and protein expression of the MAPK-pathway kinases RAS, RAF, MEK and ERK in melanoma is largely unknown. We demonstrate the impacts of antisense oligonucleotide (ASO)-based MALAT1-inhibition on MAPK-pathway gene regulation in melanoma. Our results showed that MALAT1-ASO treatment decreased BRAF RNA expression and protein levels, and MALAT1 had increased correlation with MAPK-pathway associated genes in melanoma patient samples compared to healthy skin. Additionally, drug-induced MAPK inhibition upregulated MALAT1-expression, a finding that resonates with a paradigm of MALAT1-expression presented in this work: MALAT1 is downregulated in melanoma and other cancer types in which MALAT1 seems to be associated with MAPK-signaling, while MALAT1-ASO treatment strongly reduced the growth of melanoma cell lines, even in cases of resistance to MEK inhibition. MALAT1-ASO treatment significantly inhibited colony formation in vitro and reduced tumor growth in an NRAS-mutant melanoma xenograft mouse model in vivo, while showing no aberrant toxic side effects. Our findings demonstrate new insights into MALAT1-mediated MAPK-pathway gene regulation and a paradigm of MALAT1 expression in MAPK-signaling-dependent cancer types. MALAT1 maintains essential oncogenic functions, despite being downregulated.

Abstract A010: Using oncogenic pathway agonism to sensitize RAS-mutant cancers to immunotherapy

Abstract Overall, some 30% of human cancers are driven by mutant RAS proteins, which have proven relatively difficult to target. While immunotherapy is effective for NRAS-mutant melanoma, no options exist for resistant disease, and mutant KRAS-driven lung and pancreatic carcinomas are much less responsive overall. Oncogenic pathways are often key targets for inhibition. However, pathway agonism has not been systematically explored as a therapeutic approach. BRAF-mutant melanomas respond to BRAF inhibitors (BRAFi) due to decreased ERK signaling. They can recover signaling by acquiring activating RAS/MEK mutations, but exhibit decreased proliferation in the absence of inhibitor, suggesting that supraphysiologic ERK signaling also compromises fitness. In line with this, we observed that increasing ERK hyperactivation in RAS-mutant cancers, particularly in those with a high ERK activity, might elevate ERK signaling to a degree to induce senescence and also create an inflammatory tumor microenvironment. We have shown that a majority of RAS-mutant cancer cell lines we tested undergo senescence-like arrest following exposure to all FDA-approved BRAF inhibitors at clinically relevant doses. Simultaneous MEK or ERK inhibition allows the cells to recover proliferation, and a BRAFi which does not induce ERK activation fails to have any effect, confirming that ERK hyperactivation is the key driver of this response. Importantly, these findings generalize across NRAS-mutant and KRAS-mutant cancer cell lines including in fresh human pancreatic adenocarcinoma explants. Finally, our data in two novel genomically-characterized, immunocompetent models of NRAS-mutant melanoma and KRAS-mutant pancreatic adenocarcinoma support our in-vitro findings. Both RAS-mutant murine models demonstrate that anti-PD1 therapy is substantially more efficacious when mice are additionally treated with BRAFi. Molecular profiling of the tumor microenvironment reveals the expression of cytokines related to senescence and marked infiltration of activated CD8+ T-cells. In order to implement more potentially effective immunotherapies, we have analyzed the expression of different immune-checkpoint molecules and we have observed a high expression of LAG3 and TIM3 in the melanoma NRAS-mutant model. We will further analyze the effect of those checkpoints in combination with BRAFi to potentiate the anti-tumoral responses and tumor regression. We propose that oncogenic pathway agonism is a novel, effective, and untested strategy to induce proliferation arrest and sensitization to immunotherapy. The impact of this data is in the ability to broadly sensitize RAS-mutant cancers to immunotherapy in the settings of de-novo and acquired resistance. The direct connection between a tumor cell signaling vulnerability to immunotherapy response is a major point of novelty and provides a clear molecular rationale for pursuing combined targeted and immune-based therapy. Citation Format: Alvaro de Mingo Pulido, Charles Adelmann, Brittney Sell, Karol Prieto, Kimberly Nguyen, Cynthia Dixey, Ivannie Ortiz-Rivera, Marco Napoli, Elsa Flores, Jason Fleming, Karen Mann, Christin Burd, Kenneth Y. Tsai. Using oncogenic pathway agonism to sensitize RAS-mutant cancers to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A010.