NRAS-mutant Melanoma Research Articles

237 (PB225): Targeting NOS sensitizes the NRAS-mutant melanomas to MEK inhibition and induces Immunogenic Cell Death

237 (PB225): Targeting NOS sensitizes the NRAS-mutant melanomas to MEK inhibition and induces Immunogenic Cell Death

A phase Ib study of the combination of naporafenib with rineterkib or trametinib in patients with advanced and metastatic KRAS- or BRAF-mutant non-small cell lung cancer

BackgroundGenetic alterations activating the MAPK pathway are common in non-small cell lung cancer (NSCLC). Patients with NSCLC may benefit from treatment with the pan-RAF inhibitor naporafenib (LXH254) plus the ERK1/2 inhibitor rineterkib (LTT462) or MEK1/2 inhibitor trametinib. MethodsThis first-in-human phase 1b dose-escalation/dose-expansion study investigated the combinations of naporafenib (50–350 mg once daily [QD] or 300–600 mg twice daily [BID]) with rineterkib (100–300 mg QD) in patients with KRAS-/BRAF-mutant NSCLC and naporafenib (200 mg BID or 400 mg BID) with trametinib (0.5 mg QD, 1 mg QD or 1 mg QD 2 weeks on/2 weeks off) in patients with KRAS-/BRAF-mutant NSCLC and NRAS-mutant melanoma. The primary objectives were to identify the recommended dose for expansion (RDE) and evaluate tolerability and safety. Secondary objectives included antitumor activity and pharmacodynamics. ResultsOverall, 216 patients were treated with naporafenib plus rineterkib (NSCLC: n = 101) or naporafenib plus trametinib (NSCLC: n = 79; melanoma: n = 36). In total, 10 of 62 (16%) patients experienced at least one dose-limiting toxicity. The RDEs were established as naporafenib 400 mg BID plus rineterkib 200 mg QD, naporafenib 200 mg BID plus trametinib 1 mg QD and naporafenib 400 mg BID plus trametinib 0.5 mg QD. The most frequent grade ≥ 3 treatment-related adverse event was increased lipase (8/101 [7.9%] patients) for naporafenib plus rineterkib and rash (22/115 [19.1%] patients) for naporafenib plus trametinib. Among patients with NSCLC, partial response was observed in three patients (one with KRAS-mutant, two with BRAFnon-V600-mutant NSCLC) treated with naporafenib plus rineterkib and two patients (both with KRAS-mutant NSCLC) treated with naporafenib plus trametinib. On-treatment median reductions in DUSP6 mRNA levels from baseline were 45.5% and 76.1% with naporafenib plus rineterkib or trametinib, respectively. ConclusionsBoth naporafenib combinations had acceptable safety profiles. Antitumor activity was limited in patients with NSCLC, despite the observed on-target pharmacodynamic effect.ClinicalTrials.gov identifier: NCT02974725.

613MO A phase I clinical trial evaluating exarafenib, a selective pan-RAF inhibitor in combination with binimetinib in NRAS-mutant (NRASMut) melanoma (Mel) & BRAF-altered solid tumors

613MO A phase I clinical trial evaluating exarafenib, a selective pan-RAF inhibitor in combination with binimetinib in NRAS-mutant (NRASMut) melanoma (Mel) & BRAF-altered solid tumors

Computational Modeling of Drug Response Identifies Mutant-Specific Constraints for Dosing panRAF and MEK Inhibitors in Melanoma.

This study explores the potential of pre-clinical in vitro cell line response data and computational modeling in identifying the optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential. In a drug combination screen of 43 melanoma cell lines, we identified specific dosage landscapes of panRAF and MEK inhibitors for NRAS vs. BRAF mutant melanomas. Both experienced benefits, but with a notably more synergistic and narrow dosage range for NRAS mutant melanoma (mean Bliss score of 0.27 in NRAS vs. 0.1 in BRAF mutants). Computational modeling and follow-up molecular experiments attributed the difference to a mechanism of adaptive resistance by negative feedback. We validated the in vivo translatability of in vitro dose-response maps by predicting tumor growth in xenografts with high accuracy in capturing cytostatic and cytotoxic responses. We analyzed the pharmacokinetic and tumor growth data from Phase 1 clinical trials of Belvarafenib with Cobimetinib to show that the synergy requirement imposes stricter precision dose constraints in NRAS mutant melanoma patients. Leveraging pre-clinical data and computational modeling, our approach proposes dosage strategies that can optimize synergy in drug combinations, while also bringing forth the real-world challenges of staying within a precise dose range. Overall, this work presents a framework to aid dose selection in drug combinations.

Computational modeling of drug response identifies mutant-specific constraints for dosing panRAF and MEK inhibitors in melanoma.

This study explores the potential of preclinical in vitro cell line response data and computational modeling in identifying optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential. In a drug combination screen of 43 melanoma cell lines, we identified unique dosage landscapes of panRAF and MEK inhibitors for NRAS vs BRAF mutant melanomas. Both experienced benefits, but with a notably more synergistic and narrow dosage range for NRAS mutant melanoma. Computational modeling and molecular experiments attributed the difference to a mechanism of adaptive resistance by negative feedback. We validated in vivo translatability of in vitro dose-response maps by accurately predicting tumor growth in xenografts. Then, we analyzed pharmacokinetic and tumor growth data from Phase 1 clinical trials of Belvarafenib with Cobimetinib to show that the synergy requirement imposes stricter precision dose constraints in NRAS mutant melanoma patients. Leveraging pre-clinical data and computational modeling, our approach proposes dosage strategies that can optimize synergy in drug combinations, while also bringing forth the real-world challenges of staying within a precise dose range.

Tunlametinib: First Approval.

Tunlametinib (®) is an oral, selective, mitogen-activated protein kinase kinase 1 and 2 (MEK 1/2) inhibitor being developed by Shanghai KeChow Pharma, Inc. for the treatment of solid tumours with RAS and RAF mutations, including melanoma, non-small cell cancer (NSCLC), colorectal cancer (CRC) and neurofibromatosis type 1 (NF1) plexiform neurofibromas. In March 2024, tunlametinib was granted conditional approval in China (based on surrogate endpoints) for use in patients with NRAS-mutated advanced melanoma who have failed anti-PD-1/PD-L1 treatment. This article summarizes the milestones in the development of tunlametinib leading to this first approval for the treatment of solid tumours with RAS and RAF mutations.

Tunlametinib (HL-085) plus vemurafenib in patients with advanced BRAF V600-mutant solid tumors: an open-label, single-arm, multicenter, phase I study

BackgroundTunlametinib (HL-085) is a novel, highly selective MEK inhibitor with substantial clinical activities in patients with NRAS-mutant melanoma. This phase I study evaluated the safety and preliminary efficacy of tunlametinib plus vemurafenib in patients with advanced BRAF V600-mutant solid tumors.MethodsPatients with confirmed advanced BRAF V600-mutant solid tumors who had progressed on or shown intolerance or no available standard therapies were enrolled and received tunlametinib plus vemurafenib. This study consisted of a dose-escalation phase and a dose-expansion phase. Primary end points of this study were safety, the recommended phase II dose (RP2D), and preliminary efficacy.ResultsFrom August 17, 2018 to April 19, 2022, 72 patients were enrolled. No dose-limiting toxicities occurred, and the maximum tolerated dose was not reached. The RP2D for BRAF V600-mutant non-small cell lung cancer (NSCLC) patients was tunlametinib 9 mg plus vemurafenib 720 mg, twice daily (BID, bis in die). Until the data cut-off date of December 15, 2023, of 33 NSCLC patients with evaluable disease, the objective response rate (ORR) was 60.6% (20/33; 95% confidence interval [CI], 42.1–77.1), the median progression free survival (PFS) was 10.5 months (95%CI, 5.6–14.5) and median duration of response (DoR) was 11.3 months (95%CI, 6.8-NE). At the RP2D, ORR was 60.0% (9/15; 95% CI, 32.3–83.7), the median PFS was 10.5 months (95%CI, 5.6 -NE) and median DoR was 11.3 months (95%CI, 3.9-NE). Of 24 colorectal cancer patients with evaluable disease, the ORR was 25.0% (6/24; 95% CI, 5.6-NE). All 72 patients had treatment-related adverse events (TRAEs), and the most common grade 3–4 TRAEs were anemia (n = 13, 18.1%) and blood creatine phosphokinase increased (n = 10, 13.9%). Tunlametinib was absorbed rapidly with Tmax of 0.5–1 h. Vemurafeinib did not influence the system exposure of tunlametinib and vice versa, indicating no drug-drug interaction for this combination.ConclusionsTunlametinib (HL-085) plus vemurafenib had a favorable safety profile and showed promising antitumor activity in patients with BRAF V600-mutant solid tumors. The RP2D for NSCLC was tunlametinib 9 mg BID plus vemurafeinib 720 mg BID.Trial RegistrationClinicalTrials.gov, NCT03781219.

NRAS mutation in melanoma: Association of codon-specific changes on patient outcomes.

e21545 Background: NRAS mutant melanoma is an aggressive subtype making up 20-30% of cutaneous melanoma cases without specific treatment options beyond immune checkpoint inhibition (ICI). Previous studies have suggested specific NRAS codon mutations are associated with unique signaling pathways. To test the hypothesis that NRAS codon mutations in cutaneous melanoma may drive a preferential response to ICI, we evaluated specific genetic subsets of NRAS mutant melanoma based on the Q61 and G12/G13 codons. Methods: Cutaneous melanoma samples (n = 4091) were analyzed by NGS at Caris Life Sciences. Samples were stratified by NRAS status and subdivided into NRASQ61, NRASG12/13, and NRAS-wildtype. PD-L1 expression was assessed using IHC (positive ≥ 1%). TMB-high was defined as ≥ 10 mutations/Mb. Transcriptomic signatures of MAPK pathway activation and T-cell signatures were calculated. Real-world overall survival (rwOS) information was obtained from insurance claims data, with Kaplan-Meier estimates calculated from time on ICI until last date of contact. Mann-Whitney U, Chi-square, and Fisher exact tests were applied as appropriate, with p-values adjusted for multiple comparisons. Results: Cutaneous melanoma samples were comprised of 22% NRASQ61 (n = 902), 3.1% NRASG12/13 (n = 125) and 74.8% NRASWT (n = 3064), with the most common NRAS mutation being Q61R. Patients harboring NRASG12/13 codon changes were associated with concurrent mutations in BRAF (21.0 vs 6.4%, p< 0.0001), NF1 (50.5% vs 8.7%, p< 0.0001), KIT (3.2% vs 0.2%, p< 0.001), ARID2 (22.1% vs 11.3%, p< 0.01), MAP2K1 (7.2% vs 2.6%, p = 0.022) compared to NRASQ61. NRASG12/13 tumors were more frequently TMB-high compared to NRASQ61 or NRASWT (83.1% vs 62.1% vs 55.1%, p = 0.0001), but no difference was observed in the prevalence of dMMR/MSI-H or PD-L1 expression. Transcriptomic analysis showed that NRASQ61 mutants had a significantly higher MPAS compared to NRASG12/13 or NRASWT cohorts (Median: 1.16 vs 0.67 vs 0.46, p = 0.0147), while T-cell-inflamed scores were significantly higher in NRASWT compared to NRASQ61 (Median: 4 vs -40, p = 0.00492) but not to NRASG12/13 (Median: 4 vs -13.5, p = 0.674). No difference in rwOS was identified in NRASG12/13 and NRASQ61 melanoma treated with ICI (HR: 0.753, [95% CI: 0.43 – 1.31], p = 0.316). Conclusions: Patients with NRAS-mutated cutaneous melanoma exhibit codon-specific ( NRASQ61 vs NRASG12/13) signaling and prevalence of immunotherapy-related biomarkers. Our findings indicate that melanoma samples with NRASG12/13 mutation tend to exhibit immune richness suggesting they could be viewed as a distinct group that might potentially benefit from immune therapies. This observation offers valuable insight into codon-specific tumor subsets, differential response to therapy, resistance mechanisms, and the potential development of combination therapies to enhance survival outcomes of patients with NRAS mutant melanoma.

Overall survival and efficacy subgroup analysis of tunlametinib in patients with advanced NRAS-mutant melanoma: A multicenter, open-label, single-arm, phase 2 study.

9545 Background: NRAS-mutant melanoma is an aggressive subtype with worse prognosis. However, no targeted therapy has been approved to date worldwide. Tunlametinib (HL-085) has showed an encouraging efficacy (confirmed ORR:34.8%, mPFS:4.2 months) with a manageable safety profile in phase II pivotal registrational study, which was published in ASCO 2023 annal meeting (NO.:9510). Here, we report the updated efficacy results. Methods: This is a multicenter, single-arm, phase II, pivotal registrational study. Patients (pts) with NRAS-mutant unresectable stage III or IV melanoma were enrolled and received tunlametinib 12 mg orally twice daily. The primary endpoint was confirmed objective response rate (ORR) per Response Evaluation Criteria In Solid Tumors, version 1.1 assessed by independent radiology review committee (IRRC). Results: A total of 100 pts were enrolled and 95 pts were included in the full analysis set (FAS) for efficacy analysis. At cut-off date (October 31, 2023), median follow-up was 24.4 months (95% CI: 21.9, 27.9). The confirmed ORR was 35.8% (95%CI: 26.3%, 46.3%). Median progression-free survival (mPFS) was 4.2 months (95%CI: 3.5, 5.6). The median overall survival (mOS) was immature during last year's ASCO annual meeting, and the present mOS was 13.7 months (95% CI of 10.3–18.0). The IRRC-assessed ORRs by melanoma subtype were 42.9% (95% CI of 29.7–56.8) in acral melanoma, 25.0% (95% CI of 7.3–52.4) in mucosal melanoma. ORRs by NRASmutation site were 41.3% (95% CI of 30.1–53.3), 13.3% (95% CI of 1.7–40.5) and 20.0% (95% CI of 0.5–71.6) for Q61, G12, and G13, respectively. ORR in patients who had been treated with immunotherapy was 40.6% (95% CI of 28.5–53.6), while in those without immunotherapy, ORR was 25.8% (95% CI of 11.9–44.6). The difference in PFS was not statistically significant across subgroups, regardless of melanoma type, NRAS mutation site, line of previous treatment, type of previous treatment and previous treatment with immunotherapy. The safety profile is similar with last year's ASCO annual meeting. The most frequent treatment related adverse events (TRAEs) were increased blood creatine phosphokinase (CK), diarrhea, facial oedema, peripheral oedema, and increased aspartate aminotransferase. No treatment-related death was reported. Conclusions: Tunlametinib demonstrated an encouraging treatment response rate in patients with advanced NRAS-mutant melanoma with a manageable safety profile. These results suggest that tunlametinib could be a promising treatment option for NRAS-mutant melanoma, even for those who had previously received immunotherapy. Clinical trial information: NCT05217303 .

A phase III, open-label, multicenter, randomized controlled trial of tunlametinib versus investigator-selected chemotherapy in patients with advanced NRAS-mutant melanoma who had previously received immunotherapy.

TPS9606 Background: NRAS-mutant melanoma is an aggressive subtype with worse prognosis. However, no targeted therapy has been approved to date worldwide. Tunlametinib (HL-085) has showed an encouraging efficacy (confirmed ORR:34.8%, mPFS:4.2 months) with a manageable safety profile in phase II pivotal registrational study, which was published in ASCO 2023 annal meeting (NO.:9510). Here, we present the design of phase III randomized controlled study. Methods: This is a multicenter, two-arm, open-label, randomized controlled phase III confirmatory clinical trial to evaluate the efficacy and safety of tunlametinib in comparison with the combination chemotherapy of investigator's choice in advanced NRAS-mutant melanoma patients who had previously received immunotherapy. A total of 165 subjects from about 12 sites in China will be included and randomly assigned to the corresponding treatment group in 2:1 ratio. Two stratification factors before randomization: LDH level and whether have received chemotherapy. The key inclusion criteria included: a) Patients with unresectable stage III or metastatic IV melanoma confirmed by histology or cytology; b) History of immunotherapy failure or could not tolerate immunotherapy; c) Be able to provide NRAS mutation positive test report at baseline and provide sufficient histological specimens for NRAS mutation confirmation by central laboratory. Experimental group subjects will receive continuous administration of tunlametinib 12mg BID, 28 days per cycle. Control group subjects will receive one of the following regimens which according to investigator's choice (paclitaxel plus carboplatin, or temozolomide plus cisplatin, or dacarbazine plus cisplatin) , 28 days per cycle. The two groups will receive continuous therapy until disease progression or intolerable toxicity. The primary endpoint is progression-free survival which assessed by independent radiology review committee. Secondary endpoints included overall survival, objective response rate, disease control rate, duration of response, safety, pharmacokinetics and exposure-response. Exploratory endpoints are to evaluate quality of life between two groups and to development a companion diagnostic kit for NRAS gene mutations detection. This study is currently open for enrollment. Clinical trial information: NCT06008106 .

FCN-159, a MEK1/2 inhibitor, in patients with advanced melanoma harboring NRAS or NF1mutations: A phase 1B dose-expansion study.

3095 Background: In phase 1A dose-escalation study, preliminary anti-tumor activity of FCN-159 was observed in advanced melanoma patients harboring NRAS mutations (ORR: 19.0%, mPFS: 3.8 months) with tolerable safety profile, which was posted during AACR 2022 (No.: 22-LB-7398-AACR). Here, we report the results from phase 1B dose-expansion study in advanced NRAS- or NF1-mutant melanoma patients at RP2D predetermined at 12 mg. Methods: This open-label, dose-expansion, phase 1B study (NCT03932253) consists of two cohorts, which enrolled patients with unresectable stage III/IV melanoma harboring NRAS or NF1 mutations, respectively. Patients received FCN-159 orally at 12 mg once daily (QD) continuously in 28-day cycles. The primary endpoint was anti-tumor activity. Results: As of July 17, 2023, 46 patients (32 patients with NRASmutations in Cohort 1 and 14 patients with NF1mutations in Cohort 2) were enrolled and received 12 mg FCN-159 QD. The median follow-up time was 8.6 months. Among 25 patients who failed in previous anti-PD-1 therapy in Cohort 1, 6 had confirmed partial responses (ORR, 24.0%; 95% confidence interval [CI], 9.4–45.1). Median DOR was 6.5 months (95% CI, 2.7–NE). Median progression-free survival (mPFS) was 3.6 months (95% CI, 1.8–5.5). Among 14 patients in Cohort 2, 1 had PR (ORR, 7.1%; 95% CI, 0.2-33.9) and mPFS was 2.8 months (95% CI, 1.8-5.5) (Table). Grade≥3 FCN-159-related treatment-emergent adverse events (TEAEs) were reported in 10 (21.7%) patients in both cohorts; the most common was blood creatine phosphokinase increased (13.0%). FCN-159–related serious AEs (SAEs) were reported in 3 (6.5%) patients, which were edema lower limb, cellulitis and pneumonia. Two (4.3%) patients discontinued treatment due to FCN-159 related TEAEs, including serous retinal detachment and herpes zoster. No FCN-159-related TEAEs led to death. Conclusions: FCN-159 showed encouraging efficacy data with well tolerated safety profile in patients with NRAS-mutant advanced melanoma, especially those who failed in previous anti-PD-1 therapy. Clinical trial information: NCT03932253 . [Table: see text]

Phase 1 study of ERK inhibitor JSI-1187 for patients with advanced solid tumors with MAPK pathway mutations.

e15111 Background: Mitogen-activated protein kinase (MAPK) pathway is a key regulator of cellular proliferation and survival, it is highly correlated with the occurrence of many cancers, JSI-1187 is a potent and reversible small molecule inhibitor of ERK1/2. This study is designed to assess the safety and efficacy of ERK Inhibitor JSI-1187 for advanced MAPK-mutant tumors patients. Methods: This is a phase 1 study included dose escalation and expansion (NCT06239623). The dose escalation study was conducted using a 3+3 escalation design to assess JSI-1187 16, 24, 32, 48,72, 80 and 96 mg BID, and 80, 128 and 152mg QD, repeated q 28 days. Dose limiting toxicities (DLTs) were predefined in the protocol. MAPK-mutant tumor patients whose disease progressed after existing standard treatment were treated. RAS and BRAF-mutated solid malignant tumors were explored in the dose-escalation phase. Results: As of December 23, 2023, 26 patients were enrolled, including 23 from the BID group and 3 from the 80mg QD group. No DLT was observed in QD group, 1 DLT (1 macular edema) and 2 DLTs (1 macular edema and 1 pneumonia) were observed at 96 and 80 mg BID, respectively. The most common JSI-1187 16-96mg BID related AEs were hypoalbuminemia (69.6%), rash (62.5%), and anemia (43.5%), while the most common JSI-1187 80mg QD related AEs were rash (100%), leukopenia (100%) and neutropenia (66.7%). The most common Grade 3 JSI-1187 16-96mg BID treatment-related AEs were anemia (17.4%) and thrombocytopenia (8.7%). Tumor response was evaluated in 18 of the total 26 patients. SD was noted as best response at doses of 16 – 96 mg BID and 80 mg QD including 29.8% tumor shrinkage in 1 NRAS-mutant melanoma patient treated at 32mg BID and 21.6% tumor shrinkage in 1 MEK1 K57N-mutant lung cancer patient treated at 72mg BID. Conclusions: JSI-1187 monotherapy is well-tolerated in advanced solid tumor patients and demonstrated preliminary antitumor effect. Current evidence supports future exploration of JSI-1187 as monotherapy, and in combination, in patients with MAPK-mutant tumors. Research Sponsor: JS Innopharm Clinical trial information: NCT06239623 .

αPDL2 treats melanoma in an IL17 and IFNγ dependent mechanism mediated by CD8 and γδ T cells

Abstract Immune checkpoint blockade (ICB) strategies have transformed immunotherapy with demonstrated efficacy including in melanoma. Nonetheless, most tumors do not respond to approved ICB agents. PDL2 is a relatively understudied immune checkpoint molecule that binds PD1 with higher affinity than PDL1, and also binds RGMb. We found that αPDL2 effectively treats subcutaneous B16 and Nras-mutated melanomas in aged but not young mice. αPDL2 efficacy in aged B16 requires host IL17 which induces IFNγ and is mediated by both γδ and CD8+ T cells. IL17 increased CXCR3+IFNγ+ immune cells, also supporting improved trafficking as a mechanism. αPDL2 augments B16-infiltrating γδ T cell numbers in aged mice, which boost tumor-infiltrating CD8+ T cell functions (e.g., IFNγ[TC1] ) but not numbers. CD8+ T cells did not appear to improve δγ T cell functions or numbers. αPDL2 also increased tumor infiltrating NK cells, CD4+ T cells, and dendritic cells that can mediate anti-tumor immunity. αPDL2 effectively treats heterotopic subcutaneous MB49 bladder cancer in young and aged mice showing effects are not restricted to only aged. Furthermore, lack of tumor PDL2 expression did not alter αPDL2 efficacy observed. PDL2 is thus a viable ICB target for treatment of cancer in select patient populations, including the aged.

Design, synthesis, and evaluation of novel quindoline derivatives with fork-shaped side chains as RNA G-quadruplex stabilizers for repressing oncogene NRAS translation

NRAS mutation is the second most common oncogenic factor in cutaneous melanoma. Inhibiting NRAS translation by stabilizing the G-quadruplex (G4) structure with small molecules seems to be a potential strategy for cancer therapy due to the NRAS protein's lack of a druggable pocket. To enhance the effects of previously reported G4 stabilizers quindoline derivatives, we designed and synthesized a novel series of quindoline derivatives with fork-shaped side chains by introducing (alkylamino)alkoxy side chains. Panels of experimental results showed that introducing a fork-shaped (alkylamino)alkoxy side chain could enhance the stabilizing abilities of the ligands against NRAS RNA G-quadruplexes and their anti-melanoma activities. One of them, 10b, exhibited good antitumor activity in the NRAS-mutant melanoma xenograft mouse model, showing the therapeutic potential of this kind of compounds.

Abstract CT057: Phase 1/2 trial of trametinib + ceritinib in patients with unresectable melanoma

Abstract Background: In advanced melanoma, treatment options after progression on immunotherapy are limited, particularly for patients with BRAF-WT tumors. MEK-inhibitors have been explored, with limited clinical efficacy. Our pre-clinical data demonstrated efficacy of the tyrosine kinase inhibitor ceritinib in NRAS-mutant and BRAF-mutant melanoma cells lines. There was synergy observed combining the MEK inhibitor trametinib with ceritinib (compared to trametinib alone) in BRAF-WT melanoma xenografts, with dual suppression of MAPK and mTOR signaling (Verduzco et al, Mol Cancer Ther 2018) Further, in our anti-PD-1 resistant mouse melanoma models, combination of ceritinib and trametinib was highly effective when administered subsequent to anti-PD1. (Phadke et al, Cancer Immunol Res 2021) Methods: We conducted a phase 1/2 trial of ceritinib monotherapy and then combination of trametinib with ceritinib in patients with advanced melanoma after failure of anti-PD-1 (and prior BRAF/MEK inhibitors if eligible.) Primary objective was to identify the maximum tolerated dose (MTD) of this combination, with secondary objectives of tumor response (RECIST 1.1) and progression-free (PFS) and overall survival (OS). In the initial phase 2 portion to explore single-agent activity, patients received oral ceritinib monotherapy at 450 mg daily (QD). In the subsequent phase 1 portion, patients started at dose level (DL)1 of 300 mg PO QD ceritinib + 2mg PO QD trametinib, with a BOIN design to guide dose escalation in cohort sizes of 3. DL 2 was 450 mg + 2 mg QD, while DL -1 was 300 mg + 1.5 mg QD of the combination. Tumor biopsies and blood samples were obtained on day 1 and day 15. Results: We enrolled 28 patients with unresectable/metastatic melanoma. Median age was 66, 10 female, 26 stage IV, 11 NRAS-mutant, 4 BRAF V600-mutant, with median 3 prior lines of prior therapy. There was no objective response observed to ceritinib monotherapy in 11 patients; prolonged stable disease of 6 and 8 months was seen in 2 pts. Median PFS was 2.1 months (95% CI 1.91, NA); median OS was 7.3 months (95% CI 2.4, NA). Two patients had grade 3 AST/ALT elevation; there were no unexpected toxicities. Of the 17 patients on the combination regimen, 12 received DL1. Dose-limiting toxicities of grade 3 rash and grade 2 glaucoma led to de-escalating to DL -1 for other patients. Overall, most frequent grade 3/4 toxicities were grade 3 rash (n=3), grade 3 hypertension (n=2), and grade 3 ALT/AST elevation (n=2). MTD was determined at DL -1 of 1.5 mg trametinib + 300 mg ceritinib QD. Two patients had partial response (12%), both NRAS-mutant; 6 had SD (3 lasting ≥6 months). Median PFS was 3.1 months (95% CI 1.94, 6.18) with median OS of 7.8 months (95% CI 5.1, 13.2). Overall, patients with immediate prior line of anti-PD-1 before trial treatment had a trend toward improved OS (10.2 months vs 5.1 months; p=0.21); no differences were seen based on NRAS or BRAF-status. Conclusions: Ceritinib does not appear to have meaningful single agent activity in treatment-refractory advanced melanoma. Efficacy of the combination with trametinib is modest, and the inability to dose-escalate the combination due to overlapping toxicities such as rash may have contributed. Analyses from baseline and on-treatment tumor biopsies, including immunohistochemistry and single cell RNA seq to explore drug effect on mTOR and MAPK pathways as well as immune infiltration, are ongoing and will be presented. Citation Format: Zeynep Eroglu, Vivien Yin, Joseph Markowitz, Andrew Brohl, Ahmad A. Tarhini, Wenyi Fan, Deanryan De Aquino, Ann Chen, Uwe Rix, Nikhil I. Khushalani, Keiran Smalley. Phase 1/2 trial of trametinib + ceritinib in patients with unresectable melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT057.

Assessing melanoma prognosis: the interplay between patient profiles, survival, and BRAF, NRAS, KIT, and TWT mutations in a retrospective multi-study analysis.

The incidence and prevalence of melanoma are increasing globally, presenting a significant public health concern. The main genetic drivers of melanoma include BRAF, NRAS, KIT and triple wild-type (TWT) mutations. Little is known about the effects of these mutations on outcomes in terms of demographics and patient characteristics. We examined differences in melanoma mortality risk and mutation count across mutation type and patient disease profile. We extrapolated primary melanoma patient data from 14 studies via the cBioportal database. Patients were divided into demographic groups and classified according to BRAF, NRAS, KIT and TWT mutation status. Analyses included two-sample Student t -test and two-way analysis of variance tests analysis with Tukey's post hoc test. Survival outcomes were compared via Kaplan-Meier curve and Cox regression. NRAS-mutated patients exhibited decreased overall survival compared to BRAF-mutated patients. Male patients had higher mutation counts across all gene groups than females, with the fewest TWT mutations in comparison to BRAF, NRAS and KIT mutations. Males also exhibited increased mortality risk for NRAS, KIT and TWT mutations compared to BRAF mutations. An unknown primary melanoma was associated with increased mortality risk across all gene groups. NRAS-mutated acral melanoma patients had an increased mortality risk compared to NRAS-mutated cutaneous melanoma patients. Older patients had a higher mortality risk than younger patients. Patients with heavier versus lower weights had lower mortality risk, which was more pronounced for BRAF-mutated patients. These relationships highlight the importance of demographic and pathologic relationships to aid in risk assessment and personalize treatment plans.

Targeted DNA Sequencing of Cutaneous Melanoma Identifies Prognostic and Predictive Alterations.

Cutaneous melanoma (CM) can be molecularly classified into four groups: BRAF mutant, NRAS mutant, NF1 mutant and triple wild-type (TWT) tumors lacking any of these three alterations. In the era of immune checkpoint inhibition (ICI) and targeted molecular therapy, the clinical significance of these groups remains unclear. Here, we integrate targeted DNA sequencing with comprehensive clinical follow-up in CM patients. This was a retrospective cohort study that assessed clinical and molecular features from patients with localized or metastatic CM who underwent targeted next-generation sequencing as part of routine clinical care. A total of 254 patients with CM who had a CLIA-certified targeted sequencing assay performed on their tumor tissue were included. Of the 254 patients with cutaneous melanoma, 77 were BRAF mutant (30.3%), 77 were NRAS mutant (30.3%), 47 were NF1 mutant (18.5%), 33 were TWT (13.0%) and the remaining 20 (7.9%) carried mutations in multiple driver genes (BRAF/NRAS/NF1 co-mutated). The majority of this co-mutation group carried mutations in NF1 (n = 19 or 90%) with co-occurring mutations in BRAF or NRAS, often with a weaker oncogenic variant. Consistently, NF1 mutant tumors harbored numerous significantly co-altered genes compared to BRAF or NRAS mutant tumors. The majority of TWT tumors (n = 29, 87.9%) harbor a pathogenic mutation within a known Ras/MAPK signaling pathway component. Of the 154 cases with available TMB data, the median TMB was 20 (range 0.7-266 mutations/Mb). A total of 14 cases (9.1%) were classified as having a low TMB (≤5 mutations/Mb), 64 of 154 (41.6%) had an intermediate TMB (>5 and ≤20 mutations/Mb), 40 of 154 (26.0%) had a high TMB (>20 and ≤50 mutations/Mb) and 36 of 154 (23.4%) were classified as having a very high TMB (>50 mutations/Mb). NRAS mutant melanoma demonstrated significantly decreased overall survival on multivariable analysis (HR for death 2.95, 95% CI 1.13-7.69, p = 0.027, log-rank test) compared with other TCGA molecular subgroups. Of the 116 patients in our cohort with available treatment data, 36 received a combination of dual ICI with anti-CTLA4 and anti-PD1 inhibition as first-line therapy. Elevated TMB was associated with significantly longer progression-free survival following dual-agent ICI (HR 0.26, 95% CI 0.07-0.90, p = 0.033, log-rank test). NRAS mutation in CMs correlated with significantly worse overall survival. Elevated TMB was associated with increased progression-free survival for patients treated with a combination of dual ICI, supporting the potential utility of TMB as a predictive biomarker for ICI response in melanoma.

Abstract 1753: SHOC2 is a genetic dependency in NRAS-mutant melanoma

Abstract Melanoma is the most lethal form of skin cancer, with over 300,000 new cases worldwide every year. Specifically, melanoma with NRAS mutations are of particular clinical concern due to their association with a poor prognosis and lack of specific treatment options. Therefore, there is a pressing need for novel approaches to address the treatment of NRAS-mutant melanoma. A current approach to identify novel drug targets is based on the genetic concept of induced essentiality, where functional interactions that occur in response to oncogene addiction create a dependency on another gene. To identify genetic dependencies in NRAS-mutant melanoma, whole-genome CRISPR-Cas9 knockout screens were conducted in 6 NRAS-mutant and 7 NRAS-wild-type (wt) early-passage New Zealand melanoma (NZM) cell lines that were established and maintained at 5% oxygen to mimic physiological oxygen conditions. The NZM cell lines were stably transduced with the whole-genome Brunello lentiviral single guide (sg) RNA library and screened for up to 35 days. BAGEL (Bayesian Analysis of Gene Essentiality) analyses of the NZM whole-genome knockout screens, alongside CRISPR-Cas9 screening data using the Avana sgRNA library from an additional 28 melanoma cell lines, available on the Cancer Cell Line Encyclopaedia (CCLE) database, revealed prospective gene candidates that exhibit greater detrimental effects on the fitness of NRAS-mutant cell lines compared to the NRAS-wt lines. These genes are being further validated as essential genes for NRAS-mutant melanoma cells through custom sgRNA library knockout screens and in vitro individual gene knockout studies. In particular, we demonstrate that knockout of SHOC2, a gene that encodes a scaffold protein essential for activation of the MAPK signalling pathway, results in the prevention of ERK phosphorylation and a more substantial reduction in cell growth in NRAS-mutant NZM cell lines when compared to NRAS-wt lines. These results support previous studies that have identified SHOC2 as a potential therapeutic target for RAS-driven cancers and suggest that targeting of SHOC2 may have utility in NRAS-mutant melanoma, where greater treatment options are urgently needed. Citation Format: Andrea Y. Gu, Tet-Woo Lee, Aziza Khan, Francis W. Hunter, Dean C. Singleton, Stephen M. F. Jamieson. SHOC2 is a genetic dependency in NRAS-mutant melanoma [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 1753.

Abstract 1217: Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma

Abstract Introduction: Mutations in the small GTPase NRAS are the second most common oncogenic driver in cutaneous melanoma, being found in ~25% of cases. Targeted therapy strategies are currently lacking for NRAS-mutant melanoma. Here, we characterized the anti-tumor activity and immune effects of RMC-7977, a RASMULTI(ON) preclinical tool compound, representative of the investigational agent RMC-6236, in preclinical mouse and human models of NRAS-mutant melanoma. Methods: The in vitro potency of RMC-7977 was determined using mouse and human NRAS-mutant melanoma cell lines. Recovery of signaling was quantified by Western Blot and receptor tyrosine kinase (RTK) arrays. In vivo anti-tumor activity of RMC-7977 was determined in two syngeneic mouse melanoma models (SW1 NRASG13D and NRAS#5 NRASQ61R), and tumor immune infiltrate quantified using multiplexed IHC and flow cytometry. Mechanisms of resistance were defined using RNA-seq. Results: RMC-7977 had good activity in vitro against human cancer cell lines and in mouse models of NRAS-mutant melanoma. Time-course studies demonstrated RMC-7977 rapidly inhibited phospho-ERK signaling, which then began to recover after 8 hr of treatment. There was also evidence of adaptive receptor tyrosine kinase signaling by 48 hr. In syngeneic mouse models of NRAS-mutant melanoma, RMC-7977 led to a rapid inhibition of tumor growth, increased expression of tumor MHC class I and PD-L1 and an increase in CD4+ and CD8+ T cell infiltrate into tumors. Therapeutic escape was associated with decreased CD4+ and CD8+ T cell infiltration and increased numbers of immunosuppressive MDSCs. Cells collected from tumors resistant to RMC-7977 in vivo did not show any shift in sensitivity to the inhibitor when re-challenged in vitro, suggesting that the resistance observed in vivo resulted from mechanisms specific to the tumor microenvironment, for example adaptive RTK signaling or immune escape. RNA-seq and IHC analysis suggested that resistance was associated with decreased MHC expression and antigen presentation, as well as reduced co-stimulation of T cells. Combination studies of RMC-7977 with anti-PD-1 are ongoing to identify potential synergistic interactions in these preclinical models. Conclusion: RMC-7977 has anti-tumor activity in syngeneic mouse models of NRAS-mutant melanoma, associated with a robust immune response. Citation Format: Larissa A. Carvalho, Manali S. Phadke, Keiran S. M. Smalley. Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma [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 1217.

Abstract 3008: Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma

Abstract Background: Ras oncogene activation is a common and often essential event in cancer development, including melanoma. Ras mutation is present in 20-30% of melanomas and remains an undrugged target. NRAS mutation is the most frequent Ras isoform activated in melanoma, with more than 80% cases of Q61 substitutions, and is closely associated with tumor aggressiveness. Rab25 is a ubiquitously expressed small GTPase protein with a context-dependent role in cancer pathophysiology. Its role remains undiscovered in the case of melanoma. Rab25 deep deletion can be seen in portions of melanoma in databases such as the Cancer Genome Atlas and Cbioportal. Interestingly, Rab25 has a consensus sequence homologous with the binding site of Q61 mutant Ras. We propose that the loss of Rab25 in melanocytes accompanied by Ras oncogenic activation are significant events in melanoma development, and restoration of Rab25 can potentially inhibit constitutively activated N-RAS; Rab25 serves as a tumor suppressor in melanoma genesis. Objectives: To discover the role of Rab25 in melanoma, we studied Rab25 expression via mRNA and protein levels in different melanoma cell lines and screened biopsy-proven melanoma samples through immunohistochemistry in 61 different melanoma sections. Further, to see the tumor suppressive mechanism of Rab25 in vitro, we transduced selected cell lines Rab25. Results: There is an 87% and 62.7% reduction in Rab25 mRNA level in NRASQ61R(SKMEL-2) and NRASQ61L type (WM1366) melanoma cell lines, respectively, also reflected in Rab25 protein level, and statistically significant at p-value <0.005, using student’s T-test. Further, Rab25, prominent in adjacent normal tissues, was significantly lost in biopsy-proven melanoma tissues, with an H-score ranging from 13.28 to 91.77% and 0.00 to-11.24 % for normal skin tissue and melanoma section, respectively, and a negative correlation of -0.350, indicating a promising association of Rab25 loss with the progression of melanoma. Conclusion: There is a loss of Rab25 in NRAS mutant melanoma with cancer progression. We saw a significant increase in Rab25 expression among the transduced sublines of NRAS mutant melanoma cell lines at the mRNA level, which will be further assessed through western blotting, cell proliferation assay, trans well migration assay, soft-agar colony formation assay to confirm NRAS mutant melanoma cell behavior. We will attempt to deliver a liposomally encapsulated six-peptide sequence to therapeutically inhibit the mutant NRAS. Citation Format: Samikshya Kandel, Krishna Rao. Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma [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 3008.