pan-RAF Inhibitor Research Articles

Abstract CT248: Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF- and NRAS-mutation positive solid tumors

Abstract Background: BRAFV600 alterations (BRAF Class (Cl) I alterations) encode a highly active monomeric oncogenic kinase that can be targeted by approved RAF kinase inhibitors (RAFi) in patients (pts) with melanoma (MEL), NSCLC, colorectal & anaplastic thyroid cancers. Cl II and III represent molecularly distinct BRAF alterations driving dimer-dependent signaling. Cl II alterations include single nucleotide variants (SNVs), indels and gene fusions that induce BRAF homodimer formation, leading to oncogenic kinase activation. Cl III alterations, typically SNVs, are low activity alterations that promote RAF dimerization and RAS-dependent oncogenic MAPK pathway signaling. Approved BRAFi have limited clinical activity in solid tumors driven by BRAF Cl II or III alterations, highlighting the urgency to develop effective targeted therapies for these patients. Combinations of a RAFi and a MEK inhibitor (MEKi) show promising results in early clinical trials. KIN-2787 is a potent, highly selective type II pan-RAFi designed to target both monomeric & dimeric forms of the altered BRAF kinase and minimize paradoxical activation, a liability often observed with other RAFi that can adversely impact tolerability & require addition of a MEKi to suppress pathway activation. KIN-2787 has favorable pharmaceutical properties, achieves substantial systemic exposures in pre-clinical toxicology studies, & induces regressions in human cancer xenograft models driven by BRAF Cl I, II, or III alterations. Combinations of KIN-2787 with a MEKi show promising activity in preclinical models. Methods: This is a FIH, multicenter, non-randomized Ph1 study (NCT04913285) of KIN-2787 in adult pts with BRAF mutant advanced & metastatic solid tumors (AMST). KIN-2787 will be given orally bid continuously in 28-day cycles until drug intolerance or disease progression. Planned sample size is approx. 155 pts in 2 parts: Part A is a dose-escalation to MTD open to pts with AMST driven by either BRAF genomic alterations (Cl I, II or III ) or by NRAS. Part A1 assesses single agent KIN-2787; Part A2 assesses KIN-2787 in combination w/binimetinib. Part B will evaluate a selected dose of KIN-2787 in 3 cohorts of pts (MEL, NSCLC, or other AMST), each driven by BRAF Cl II or III alterations. Standard Ph1 enrollment criteria are required (ECOG PS 0-2, normal organ function, prior receipt of standard treatment or medical judgment that such is not appropriate). Pts may have measurable or evaluable disease. Key exclusion criteria include known active brain metastases, prior BRAF-, MEK-, or MAPK inhibitor therapy (unless for FDA approved indications), HBV/HCV seropositivity. Primary endpoints are safety/tolerability (Part A), and preliminary antitumor activity: objective response rate, disease control rate, duration of response, & duration of stable disease (Part B). Secondary objectives include pharmacokinetic and pharmacodynamic assessments including measures of MAPK signaling inhibition. Enrollment began in August 2022. Trial is open globally. Citation Format: Meredith McKean, Cesar A. Perez, Shumei Kato, Alexander I. Spira, Yan Xing, Hao Xie, Michael Millward, Anthony Olszanski, Ken Kobayashi, Nichol Miller, Paul Severson, Betty Tam, Caro Unger. Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF- and NRAS-mutation positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT248.

Abstract 2677: JZP815, a potent and selective pan-RAF inhibitor, demonstrates efficacy in RAF and RAS mutant tumor pre-clinical models

Abstract Activation of the mitogen-activated protein kinase (MAPK) pathway by oncogenic mutations in RAS and RAF is a frequent driver of human cancer. Targeting specific components of the pathway is a precise and rational route to deliver benefits to cancer patients with high unmet therapeutic needs. The RAF kinase proteins (ARAF, BRAF, CRAF) are core pathway components which mediate MAPK signaling. Mutations in these critical signaling proteins leads to constitutive activation of the MAPK pathway and tumor growth. Using state of the art screening methodologies and medicinal chemistry we discovered and developed a next generation pan-RAF kinase inhibitor, JZP815, which potently inhibits wild-type and mutant RAF kinases in cell-free and cell-based assays. JZP815 is a selective RAF kinase inhibitor that suppresses all 3 RAF kinase family members at low-to-sub nanomolar potencies in cell-free assays. Moreover, in tumor cells JZP815 does not induce significant paradoxical pathway activation, observed with approved 1st generation BRAF-selective inhibitors, while demonstrating equivalent potencies for MAPK pathway inhibition driven by either mutant RAF monomers or RAS-induced RAF dimers in tumor cells. JZP815 significantly inhibited tumor growth as a single agent in multiple mouse xenograft solid tumor models harboring RAS and/or BRAF mutations. Furthermore, JZP815’s favorable pharmacokinetic profile sustained on-target pathway pharmacodynamic responses in a predictable dose and time dependent manner. JZP815 demonstrated enhanced activity when combined with inhibitors of other MAPK pathway components in both class 2 and class 3 mutant BRAF patient-derived tumor cells ex vivo, and KRAS mutant NSCLC and CRC xenografts in vivo. In summary, JZP815 is a potent and selective pan-RAF inhibitor that is orally bioavailable in multiple preclinical species with a well-behaved safety profile and pharmacokinetic properties predicted to provide drug exposure required for target engagement in humans, and therefore suitable for advancing into first-in-human trials. Citation Format: Robert Hauptschein, Simon Woodcock, Andrew Belfield, Helen Mason, Dorottya Keppel, Svetlana Markova, Kyuri Kim, Shane Roller, Caroline Phillips, Clifford D. Jones, Robin C. Humphreys. JZP815, a potent and selective pan-RAF inhibitor, demonstrates efficacy in RAF and RAS mutant tumor pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2677.

Abstract 4196: A controlled drug release device facilitates the identification of favorable combinations of immune-oncology drugs with targeted or cytotoxic drugs in a patient-derived humanized mouse model of renal cancer

Abstract Identifying how to optimally combine immunotherapies with other available anti-cancer therapies is a major challenge in oncology. We have utilized an implantable microdevice performing cassette microdosing that measures intratumor drug responses and anti-tumor immunity for eleven agents in parallel. Local tumor response is measured by cyclical immunofluorescence for deep cellular response phenotyping. This approach is combined with the systemic administration of checkpoint inhibitors to examine whether local immunogenic cell death (ICD) induced by a given drug microdose potentiates the immunotherapy’s anti-tumor effect. The measurements were performed in a humanized mouse model of clear cell renal cancer, patient derived xenograft (PDX) RXF488. RXF488 was implanted subcutaneously in 30 NSG mice. Animals were stratified into 6 groups with n= 4-6. Humanization was performed by the intravenous injection of 5x10e6 human peripheral blood mononuclear cells prior to the first treatment. Systemic anti-PD1 treatment was applied in the presence and absence of the microdevice loaded with eleven different drugs. Control groups received the empty microdevice in the presence or absence of PBMC. Beside the histological examination of the tumor tissue, flow cytometry (FC) was performed on bone marrow, spleen and tumor tissue to determine infiltration of human immune cells. FC analyses revealed no influence of the treatment on the human immune cells in bone marrow and spleen. The anti-PD1 treatment induced an increase in huCD45+ cells specifically in the tumor tissue only 48h after treatment. Several agents showed a significant increase in apoptosis induction when nivolumab was added: The largest increase was observed for the panRAF inhibitor LXH254, Sorafenib, Oxaliplatin and Doxorubicin. The increased efficacy from immunotherapy administration has a strong positive correlation with increased induction of ICD in the tumor microenvironment determined by CD11b, ICAM-1 and MHC-II expression: drugs that showed the highest increase in apoptosis when combined with aPD-1 showed an increased likelihood for markers associated with ICD, namely Oxaliplatin and LXH254. We were able to verify the results of the screening experiment in a conventional setting with systemic combination treatment arms in the same PDX model. Our results demonstrate that local tumor response signatures of ICD can be used to systemically identify synergistic combinations of a range of drugs with immunotherapy on a tumor specific basis. The approach may represent a new paradigm for efficient in vivo screening of novel combinations, particularly with combinations involving immunotherapies. Citation Format: Oliver Jonas, Eva Oswald, Kanstantsin Lashuk, Sebastian Ahn, Julia Schüler. A controlled drug release device facilitates the identification of favorable combinations of immune-oncology drugs with targeted or cytotoxic drugs in a patient-derived humanized mouse model of renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4196.

OTHR-18. A pilot study evaluating the access, utilization, and patient satisfaction of basic skin care products among pediatric patients prescribed medications that target the mitogen-activated protein kinase pathway

BACKGROUND: The mitogen-activated protein kinase (MAPK) pathway is dysregulated in approximately one third of all malignancies. Agents targeting this pathway are known to cause frequent and significant skin toxicity. METHODS: Patients <21 years of age prescribed any medication targeting the MAPK pathway within the past six months were eligible for this prospective, pilot study. Each participant received a complimentary skin care kit consisting of basic skin and oral care items. At baseline and three months after enrollment, participants completed surveys assessing demographics, skin symptoms, use of the products, and quality of life (QOL; Dermatology Life Quality Index and Child Dermatology Life Quality Index, CDLQI). OBJECTIVES: Primary aims were evaluating utilization of the kit and measuring patient satisfaction of the items included. Secondary aims included determining skin-related QOL and assessing adherence to MAPK targeted therapy. RESULTS: Eleven participants (seven male, four female; median age 13 years, range 4-21 years) have enrolled to date (enrollment goal of 30). Participant therapies included MEK inhibitors (nine), a pan-RAF inhibitor (one), and combination MEK-mTOR inhibition (one). Six have completed the three-month follow-up. Of these six, four rated their satisfaction with the kit as excellent, one each rated very good and fair satisfaction. Five participants rated the kit helpful and thought the kits should be distributed when patients begin therapy. Three started taking their targeted agent before enrolling in the study and their CDLQI changed from 3 to 0.3 (means; improved). Three participants started their targeted agent after enrollment and their CDLQI changed from 1 to 3.3 (means; worsened). CONCLUSIONS: Participants reported that skin care kits were helpful and recommended use by other patients on MAPK-targeted agents. Continued follow-up and enrollment in the study will help further explore the utility of specific products in the kits, as well as changes in patients’ QOL.

Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in combination with MEK inhibition in preclinical models of human NRAS mutant melanoma.

e15099 Background: NRAS mutations which activate MAPK signaling represent oncogenic driver alterations in approximately 20% melanoma cases in the US. NRAS mutant melanomas are uniquely dependent upon CRAF rather than BRAF for activation of downstream MEK/ERK signaling. In BRAF mutant melanoma, approved RAF-targeted therapies are commonly used in combination with a MEK inhibitor which provides clinical benefit by inhibition of two targets within the oncogenic MAPK signaling pathway. Emerging data with pan-RAF inhibitors in early clinical development suggests benefit with and without combined MEK inhibition, yet no approved targeted therapy exists for NRAS mutant melanoma patients. KIN-2787 is a novel, orally available pan-RAF inhibitor designed to be effective in RAF-dependent cancers, regardless of isoform. Methods: Kinome profiling was evaluated by radiometric enzyme assay at Reaction Biology across 688 kinases (including wild type, atypical, and mutant). Cellular activity was assessed by suppression of downstream MAPK pathway signaling and cell growth inhibition in human tumor cell lines. Combination cell growth inhibition studies were performed in 9x5 dose matrices with KIN-2787 and binimetinib, respectively. Extended cell growth inhibition effects were assessed by Incucyte imaging. In vivo KIN-2787 and combination efficacy was evaluated in NRAS mutant xenograft models. Results: Kinome profiling of KIN-2787 revealed exquisite selectivity with only 2 of 669 non-RAF family kinases inhibited &gt; 75% at 1 M KIN-2787 and retained ̃10x and 70x selectivity window against those two kinases, DDR1 and p38b, respectively, relative to RAF kinases. We previously reported KIN-2787 activity across BRAF, NRAS, and KRAS mutant tumor cell lines with greatest sensitivity in Class II and III dimer-driven BRAF models. Here, we evaluated NRAS mutant, BRAF WT melanoma for combination potential with binimetinib. Melanoma tumor cell lines bearing NRAS hotspot mutations demonstrated synergistic benefit with KIN-2787 combined with binimetinib. Daily KIN-2787 plus binimetinib treatment in NRAS mutant melanoma xenograft models resulted in significant tumor growth inhibition benefit relative to either agent alone and was associated with added MAPK pathway biomarker suppression. Conclusions: KIN-2787 is a highly selective, potent, next-generation, pan-RAF inhibitor with activity across BRAF and RAS mutant human tumor cell models. Preclinical in vitro and in vivo studies using KIN-2787 in combination with binimetinib demonstrated significant combination benefit in NRAS mutant melanoma models. Taken together with its unique selectively, these data support use of KIN-2787 in combination therapy in this patient segment. A Phase 1/1b dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285).

FIREFLY-1 (PNOC 026): A phase 2 study to evaluate the safety and efficacy of tovorafenib (DAY101) in pediatric patients with RAF-altered recurrent or progressive low-grade glioma or advanced solid tumors.

TPS10062 Background: RAF gene fusions ( BRAF and RAF1) and BRAF V600E mutations are oncogenic drivers found on a mutually exclusive basis in most pediatric low-grade gliomas (LGGs). In addition, RAF fusions ( BRAF and RAF1) have also been identified in other pediatric solid tumors. Tovorafenib (DAY101) is an investigational, oral, highly selective, CNS-penetrant, small molecule, type II pan-RAF inhibitor. In contrast to type I BRAF inhibitors, tovorafenib does not induce RAS-dependent paradoxical activation of the MAPK pathway. In the phase 1 PNOC014 study in pediatric patients with recurrent/progressive LGG, tovorafenib was well tolerated and 7/8 patients with tumor harboring RAF fusions had meaningful clinical benefit. Recently, a child with a novel SNX8-BRAF fusion spindle cell sarcoma demonstrated a rapid and deep response when treated with tovorafenib. Methods: FIREFLY-1 (NCT04775485) is an open-label, multicenter, phase 2 study evaluating the safety and efficacy of tovorafenib monotherapy in pediatric patients with RAF-altered recurrent or progressive LGG or advanced solid tumors. The initial design included only patients with LGG (arm 1). Two new arms have now been added; arm 2 will allow tovorafenib treatment for patients with LGG harboring an activating RAF alteration after completion of enrollment to arm 1 and prior to tovorafenib regulatory approval; arm 3 will enroll patients with advanced solid tumors harboring an activating RAF fusion. Eligible patients are 6 months to 25 years of age, who have received ≥1 prior line of systemic therapy with documented radiographic progression, have evaluable and/or measurable disease by appropriate criteria, a Karnofsky or Lansky performance score of at least 50, and adequate organ function. Patients are excluded if their tumor has other driver mutations, they have neurofibromatosis type 1, central serous retinopathy, retinal vein occlusion, clinically significant active cardiovascular disease, or are currently being treated with a strong CYP2C8 inhibitor or inducer other than those allowed per protocol. Approximately 140 patients in total will be enrolled including 60 in arm 1, 60 in arm 2 and 20 in arm 3. Tovorafenib will be administered at 420 mg/m2 (not to exceed 600 mg) weekly (days 1, 8, 15 and 22) for 26, 28-day cycles (in the absence of disease progression or unacceptable toxicity). They may then continue tovorafenib or enter a drug holiday period. The primary endpoint is overall response rate, as defined by the RANO criteria (arm 1) or RECIST v1.1 (arm 3) and as determined by an independent radiology review committee. Secondary endpoints (arms 1 and 3) include safety and tolerability, pharmacokinetics, duration of response, time to response and progression-free survival. Tovorafenib is available in tablet or liquid suspension formulations. Clinical trial information: NCT04775485.

Classical RAS proteins are not essential for paradoxical ERK activation induced by RAF inhibitors

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.

Vertical Inhibition of the RAF-MEK-ERK Cascade Induces Myogenic Differentiation, Apoptosis, and Tumor Regression in H/NRASQ61X Mutant Rhabdomyosarcoma.

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF-MEK-ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF-MEK-ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.

Abstract P154: Genomic profiling and matched therapy for recurrent or metastatic salivary gland neoplasms. Results from the matched cohort of the GEMS-001 clinical trial

Abstract Background Salivary gland tumors (SGT) are rare and heterogeneous diseases with limited systemic options available. Results from recent basket trials including SGT cohorts for specific actionable alterations (AA) have described promising outcomes with molecularly targeted approaches (TA). Methods The GEMS-001 study (NCT02069730) is an ongoing multi-arm trial with two phases. In part 1, recurrent or metastatic (R/M) SGT patients (pts) undergo comprehensive immunohistochemistry (IHC) including HER2, androgen receptor (AR) and ALK translocation and multigene panel testing (NGS). Based on the molecular profiling results in part 1, R/M SGT pts are matched to available targeted therapies on disease progression. Here, we present the efficacy results for the matched cohort of the GEMS-001 study. Results As of July 2021, 100 pts underwent molecular profiling within part 1. According to the SGT subtype (WHO 2017): 49% adenoid cystic carcinoma (ACC), 18% salivary duct carcinoma (SDC), 9% acinic (ACI) and 24% other subtypes (O). Overall, on IHC HER2 overexpression was present in 10%, AR 24% and ALK translocation 0%. On NGS, PIK3CA mutation (mut) was in 10%, HRAS mut 7%, ERBB2/3 alterations (alt) 5%, NOTCH1-3 mut 2% and ETV6-NTRK3 fusion 2%. Up to 45% pts displayed at least 1 AA and 25% had ≥2 AA. AA were enriched in SDC (100% ≥1 AA) as compared to ACI (33% ≥1 AAs), O (58% ≥1 AA) or ACC (22% ≥1 AA) (p&amp;lt;0.001 Fisher's exact test). A total of 34 pts (34%) were matched to proteomic or genomic alterations. Of those matched, median age was 62 years (range 47-84), M:F 26:8, 100% ECOG≤1, and their AA-TA included 15 AR (leuprolide+bicalutamide), 4 HER2 or ERBB2 alt (trastuzumab+pertuzumab), 4 PIK3CA mut (PIK3CA inhibitor), 2 cyclin pathway alt (palbociclib), 2 ETV6-NTRK3 fusion (larotrectinib) and 7 other AAs (1 EGFR L858 mut (afatinib), 1 c-KIT mut (imatinib), 1 BAP1 mut (olaparib), 1 Non-V600 BRAF mut (pan-RAF inhibitor), 1 CHEK2 mut (olaparib), 1 ATM mut (olaparib) and 1 PTCH1 (vismodegib)). As per efficacy, overall response rate was 24% and median progression free survival 6.4 months (m) (95%CI 3.4-8.9 m) for the matched population. Complete responses were observed in 2 pts treated with leuprolide+biculatamide (AR). Partial responses were observed in 2 pts treated with larotrectinib (ETV6-NTRK3), 3 pts treated with trastuzumab+pertuzumab (HER2) and 1 pt treated with leuprolide+biculatamide (AR). Notably, among responders median duration of response was 13.9 m (95%CI 4.6-18.0 m). Conclusions In our cohort, more than one third of the population were matched to TA with promising efficacy outcomes. Our results support comprehensive molecular and IHC profiling but its clinical utility may vary depending on the SGT subtype. Citation Format: Alberto Hernando-Calvo, Eoghan Malone, Daphne Dai, Amy Prawira, Ilan Weinreb, Anneli Eliason, Angela Rodriguez, Katherine Lajkosz, Sarah Jennings, Anna Spreafico, Lillian L. Siu, Aaron Hansen. Genomic profiling and matched therapy for recurrent or metastatic salivary gland neoplasms. Results from the matched cohort of the GEMS-001 clinical trial [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P154.

Abstract P226: Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF mutation positive solid tumors

Abstract Background BRAFV600 mutations (BRAF Class I mutations) encode a highly active monomeric oncogenic kinase that can be targeted by approved RAF kinase inhibitors in patients (pts) with melanoma (MEL), NSCLC, colorectal &amp; anaplastic thyroid cancers. In other discrete patient populations, molecularly distinct BRAF alterations drive dimer-dependent oncogenic signaling including BRAF Class II and III alterations. Class II alterations include single nucleotide variants (SNVs), indels and gene fusions that induce BRAF homodimer formation leading to oncogenic kinase activation. Class III mutations, typically SNVs, are low activity kinase mutations that promote RAF dimerization and oncogenic MAPK pathway signaling that is RAS-dependent. Approved BRAF inhibitors have limited clinical activity in diverse solid tumors driven by BRAF Class II or III alterations, highlighting the urgency to develop effective targeted therapies for these patients. KIN-2787 is a potent, highly selective pan-RAF type II kinase inhibitor designed to target both monomeric &amp; dimeric forms of the mutant BRAF kinase and minimize paradoxical activation, a liability often observed with other RAF inhibitors that can adversely impact tolerability &amp; require addition of a MEK inhibitor to suppress pathway activation. KIN-2787 has favorable pharmaceutical properties, achieves substantial systemic exposures in pre-clinical toxicology studies &amp; induces regressions in human cancer xenograft models driven by BRAF Class I, II or III mutations. Methods This is a FIH, multicenter, non-randomized, open-label, Ph1 study (NCT04913285) of KIN-2787 in adult patients with BRAF mutant advanced &amp; metastatic solid tumors (AMST). KIN-2787 will be given orally bid continuously in 28-day cycles until drug intolerance or disease progression. Planned sample size is approx. 115 pts in two parts: Part A is a dose-escalation to MTD open to pts with AMST driven by BRAF Class I, II or III genomic alterations. Part B will evaluate a selected dose of KIN-2787 in 3 cohorts of pts with MEL, NSCLC, or other AMST, each driven by BRAF Class II or III alterations. Standard Ph1 enrollment criteria are required (ECOG PS ≤ 2, normal organ function, prior receipt of standard treatment or medical judgment that such is not appropriate). Patients may have measurable or evaluable disease per RECIST v1.1. Key exclusion criteria include known active brain metastases from non-brain tumors, prior receipt of BRAF-, MEK-, or MAPK-directed inhibitor therapy (unless for FDA approved indications), HBV &amp; HCV seropositivity. Primary endpoints are safety/tolerability (Part A), and preliminary antitumor activity: objective response rate (ORR), disease control rate (DCR), duration of response (DOR), &amp; duration of stable disease (Part B). Secondary objectives include pharmacokinetic (PK) and pharmacodynamic (PD) assessments including tissue and blood-based measures of MAPK signaling inhibition. This study recently opened for Part A recruitment in the United States (NCT04913285). Citation Format: Meredith McKean, Shumei Kato, Alexander Spira, Yan Xing, Hao Xie, Elizabeth Buchbinder, Ken Kobayashi, Richard Williams. Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF mutation positive solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P226.

High-throughput ex vivo drug testing identifies potential drugs and drug combinations for NRAS-positive malignant melanoma

Therapy options for patients with metastatic melanoma (MM) have considerably improved over the past decade. However, many patients still need effective therapy after unsuccessful immunotherapy, especially patients with BRAF-negative tumors who lack the option of targeted treatment second line. Therefore, the elucidation of efficient and personalized therapy options for these patients is required. In this study, three patient-derived cancer cells (PDCs) were established from NRAS Q61-positive MM patients. The response of PDCs and five established melanoma cell lines (two NRAS-positive, one wild type, and two BRAF V600-positive) was evaluated toward a panel of 527 oncology drugs using high-throughput drug sensitivity and resistance testing. The PDCs and cell lines displayed strong responses to MAPK inhibitors, as expected. Additionally, the PDCs and cell lines were responsive to PI3K/mTOR, mTOR, and PLK1 inhibitors among other effective drugs currently undergoing clinical trials. Combinations with a MEK inhibitor were tested with other targeted agents to identify effective synergies. MEK inhibitor showed synergy with multikinase inhibitor ponatinib, ABL inhibitor nilotinib, PI3K/mTOR inhibitor pictilisib, and pan-RAF inhibitor LY3009120. The application of the patients’ cancer cells for functional drug testing ex vivo is one step further in the process of identifying potential agents and agent combinations to personalize treatment for patients with MM. Our preliminary study results suggest that this approach has the potential for larger-scale drug testing and personalized treatment applications in our expansion trial. Our results show that drug sensitivity and resistance testing may be implementable in the treatment planning of patients with MM.

RAF1 amplification: an exemplar of MAPK pathway activation in urothelial carcinoma.

Despite recent therapeutic gains in the treatment of advanced bladder cancer, the overall survival in patients with metastatic disease remains poor and further therapeutic discovery is needed. Advanced bladder cancer is a molecularly heterogeneous disease, and the identification of driver genetic alterations has led to effective targeted therapeutic agents, such as fibroblast growth factor receptor (FGFR) inhibitors. In this issue of the JCI, Bekele et al. identify a subtype of muscle-invasive bladder cancer (MIBC) that harbors RAF1 amplification. The authors showed that RAF1 inhibition, with pan-RAF inhibitors, and the combination of RAF1 inhibition with MEK inhibition were efficacious in preclinical models harboring RAF1 amplifications as well as in tumors with HRAS and NRAS mutations. This study highlights RAF1 amplification as a driver event in bladder cancer and establishes the central role of the MAPK pathway in bladder tumorigenesis.

Exploiting LY3009120 and Asciminib Combination to Target TKI-Resistant CML

The oncogenic BCR-ABL1 tyrosine kinase is the driver of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Tyrosine kinase inhibitors (TKIs) targeting ABL kinase are generally effective, but subsets of patients treated with single-agent TKIs develop resistance due to mutations in BCR-ABL1 that impair TKI binding. We have previously reported that BCR-ABL1 compound mutants (exhibiting two mutations within the same BCR-ABL molecule) that include the T315I gatekeeper mutation confer a high degree of resistance to all clinical ABL TKIs used as single agents, including ponatinib and the allosteric inhibitor asciminib. However, combining asciminib with ponatinib provides an effective strategy for overcoming compound mutation-based resistance (Eide et al. Cancer Cell 2019). As the clinical utility of ponatinib is limited by cardiovascular toxicity, including arterial occlusive events (AOEs), we decided to search for alternative molecules for use in combination with asciminib.To identify functional ponatinib analogs, we performed Quantum Similarity Modeling (QSM) on the reported crystal structure of T315I mutant ABL1 kinase in complex with nilotinib and asciminib (5MO4) (Wylie et al. Nature 2017) to search for other molecules. Compared to conventional computational modeling, QSM identifies novel classes of structurally distinct compounds that are comparable on a quantum level by precisely defining their interaction with the target. Affinity inferred by close complementarity with the shared ligand-protein surface in the region of the surveyed binding site is mapped, using multiple weak local associations. Our in silico QSM platform combines quantum methods with machine learning to investigate extensive chemical spaces. We screened several million compounds against BCR-ABL1 and identified 51 potential candidates predicted effectively to block T315I mutant BCR-ABL1 when combined with asciminib. To prioritize potent and non-toxic drug combinations for further development against compound mutants, we initially profiled all 51 compounds for their efficacy against Ba/F3 BCR-ABL T315I cells, alone and in combination with asciminib (1 nM). Of 51 compounds, LY3009120, a pan-RAF inhibitor that is currently in phase I clinical development for advanced solid malignancies (Sullivan et al. Mol Cancer Ther 2020), showed strong activity against BCR-ABL T315I when combined with asciminib. These data provided proof of principle for the QSM approach.We next tested the efficacy of all 51 candidates ± asciminib against Ba/F3 cells harboring T315I-inclusive BCR-ABL1 compound mutants, including Y253H/T315I, E255V/T315I, H396R/T315I, G250E/T315I, and T315L as the most resistant mutants. Neither single agent showed any effect. However, LY3009120 strongly inhibited BCR-ABL1 compound mutants when combined with asciminib. No toxicity was observed against Ba/F3 parental cells, confirming that the effects of the combinations are mediated by inhibition of BCR-ABL1. Synergy quantification of the dose-response matrix for the LY3009120/asciminib combination using the Zero Interaction Potency model demonstrated highly synergistic interactions (Synergy score > 10) between the two inhibitors. To directly assess the binding affinity of LY3009120 to the ABL1 kinase domain, we used the cell-based NanoBRET intracellular ABL1 kinase assay on HEK-293 cells expressing luciferase-tagged ABL1. The NanoBRET assay uses energy transfer to quantify the affinity of test compounds by competitive displacement of a cell-permeable fluorescent tracer that is reversibly bound to an ABL1-luciferase fusion protein. We found that LY3009120 competes off the fluorescent tracer at a low micromolar range (EC 50 = 0.75 μM), confirming direct binding of LY3009120 to the kinase domain of ABL1. We hypothesize that the binding of LY3009120 to the ABL1 kinase domain induces a conformational change that re-establishes asciminib binding to the myristoyl binding pocket, allowing for synergy. Studies to quantify the binding affinity of LY3009120 and asciminib to BCR-ABL1 mutants are underway, and data will be presented.In summary, our findings validate QSM as a novel in silico approach to identify TKI combinations. Combining LY3009120 with asciminib may be an effective, low-risk strategy to target BCR-ABL1 compound mutants in patients with clinical TKI resistance. DisclosuresDeininger: SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Novartis: Consultancy, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding.

Anti-tumor activity of the pan-RAF inhibitor TAK-580 in combination with KPT-330 (selinexor) in multiple myeloma.

RAS/RAF/MEK/ERK pathway inhibitors exhibit significant anti-tumor effects against various tumor types, including multiple myeloma (MM), and they are predicted to play a pivotal role in precision medicine. The XPO1 inhibitor KPT-330 has also exhibited promising efficacy in combination with other novel drugs in treating relapsed/refractory MM (RRMM). In this study, we explored the anti-tumor effects of a combination of the pan-RAF inhibitor TAK-580 and KPT-330. Importantly, TAK-580 enhanced KPT-330-induced cytotoxicity and apoptosis in human myeloma cell lines and primary myeloma cells from RRMM patients. Moreover, TAK-580 and KPT-330 synergistically inhibited nuclear phospho-FOXO3a and enhanced cytoplasmic phospho-FOXO3a in MM cells, leading to cytoplasmic enhanced Bim expression and finally apoptosis. This indicates that TAK-580 enhances KPT-330-induced cytotoxicity and apoptosis primarily via the FOXO3a-Bim axis. In addition, TAK-580 enhanced the cytotoxicity of KPT-330 against MM cells even in the presence of IGF-1. Taken together, our results demonstrate that a combination of pan-RAF inhibitor and XPO1 inhibitor is a potential therapeutic option in treating MM.

An Efficient Second-Generation Manufacturing Process for the pan-RAF Inhibitor Belvarafenib

Herein, the development of a streamlined manufacturing process for the pan-RAF inhibitor belvarafenib (GDC-5573) is reported. The process to belvarafenib features a number of efficient key reactions, including a robust and scalable Pd-catalyzed carbonylation reaction to generate thienopyrimidine 2 and a highly chemoselective Pt/V/C-catalyzed nitro group reduction to access the penultimate intermediate 3. The final amide coupling was accomplished by a mild and safe protocol employing N,N,N′,N′-tetramethylchloroformamidinium hexafluorophosphate as the coupling reagent, which afforded belvarafenib on a multikilogram production scale after recrystallization.

Novel insights into the pathogenesis and treatment of NRAS mutant melanoma

ABSTRACT Introduction NRAS was the first mutated oncogene identified in melanoma and is currently the second most common driver mutation in this malignancy. For patients with NRASmutant advanced stage melanoma refractory to immunotherapy or with contraindications to immune-based regimens, there are few therapeutic options including low-efficacy chemotherapy regimens and binimetinib monotherapy. Here, we review recent advances in preclinical studies of molecular targets for NRAS mutant melanoma as well as the failures and successes of early-phase clinical trials. While there are no targeted therapies for NRAS-driven melanoma, there is great promise in approaches combining MEK inhibition with inhibitors of the focal adhesion kinase (FAK), inhibitors of autophagy pathways, and pan-RAF inhibitors. Areas Covered This review surveys new developments in all aspects of disease pathogenesis and potential treatment – including those that have failed, stalled, or progressed through various phases of preclinical and clinical development. Expert Opinion There are no currently approved targeted therapies for BRAF wild-type melanoma patients harboring NRAS driver mutations though an array of agents are in early phase clinical trials. The diverse strategies taken exploit combined MAP kinase signaling blockade with inhibition of cell cycle mediators, inhibition of the autophagy pathway, and alteration of kinases involved in actin cytoskeleton signaling. Future advances of developmental therapeutics into late stage trials may yield new options beyond immunotherapy for patients with advanced stage disease and NRAS mutation status.

Abstract 1481: DAY101, a potent pan-RAF inhibitor with activity in preclinical models harboring MAPK pathway alterations beyond BRAF V600E mutation

Abstract DAY101 (formerly TAK-580) is an oral, CNS-penetrant, selective small molecule pan-RAF kinase inhibitor that is equipotent against BRAF V600E, wildtype BRAF, and CRAF and can inhibit both RAF monomers and dimers, including BRAF fusions. KIAA1549:BRAF fusions have been identified as a key oncogenic driver for pilocytic astrocytoma (Jones et al. 2008). DAY101 has demonstrated significant activity against this fusion preclinically (Sun et al. 2017). Clinical data for DAY101 has shown a durable 63% response rate with good tolerability in children with low-grade glioma (LGG) harboring RAF fusions (Wright et al. 2020). To explore the potential impact of DAY101 in adult solid tumors harboring specific genomic alterations beyond BRAF V600E, a panel of tumor cell lines and PDX models harboring BRAF fusions, non V600 BRAF mutations or RAS mutations were assessed for anti-proliferative activity in response to DAY101. Furthermore, hypothesis-driven MAPK pathway node inhibitor combinations were explored for potential synergy. While performing favourably in preclinical in vivo studies, DAY101 does exhibit some technical challenges in long-term in vitro assays, particularly in those that extend for more than three days. Accordingly, the tool compound TAK-632, which exhibits comparable biochemical and cellular potency to DAY101, was used alternatively as needed for in vitro assays. Single-agent anti-proliferative activity was observed in a melanoma BRAF fusion PDX model treated with TAK-632 ex vivo, with less sensitivity observed in models beyond melanoma harboring other BRAF fusions. Ongoing combination studies with MEK inhibitors demonstrated strong synergy in PDX models harboring BRAF fusions ex vivo, as assessed by Bliss independence or other relevant scoring systems. In cell lines or PDX models harboring non V600 BRAF mutations, in vitro combination studies demonstrated strong synergy when DAY101 was combined with MEK inhibitors, as assessed by Bliss independence score. DAY101 exhibited modest single agent anti-proliferative activity in a panel of KRAS mutant cell lines in vitro and ongoing combination studies will assess potential synergy when combined with MAPK pathway node inhibitors. DAY101 is currently in clinical development for the treatment of patients under 25 years old with LGG harboring RAF alterations, including BRAF fusions and BRAF mutations. Inhibition of both RAF monomers and dimers by DAY101 enables a broader indication selection strategy, beyond LGG and BRAF alterations. Ongoing translational work will highlight the potential utility of DAY101 in adult tumors harboring RAS or RAF alterations, both as a single agent and in hypothesis-driven combinations. Citation Format: Eleni Venetsanakos, Mike Preigh, Jeannie Hou, Michael C. Cox, Jeremy Bender, Sam C. Blackman. DAY101, a potent pan-RAF inhibitor with activity in preclinical models harboring MAPK pathway alterations beyond BRAF V600E mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1481.

The next-generation pan-RAF inhibitor, KIN-2787, is active in class II and class III BRAF mutant models.

3116 Background: Oncogenic BRAF gene alterations, leading to aberrantly activated BRAF monomers (Class I mutations) or dimers (Class II and Class III mutations), are observed in approximately 6% of all human cancers. First-generation BRAF inhibitors targeting Class I BRAF mutants, including dabrafenib, encorafenib, and vemurafenib, provide significant clinical benefit to patients with BRAF V600 mutation-driven melanoma and select solid tumors as monotherapies or in combination with other targeted therapies. The currently approved BRAF inhibitors have not, however, proven to be effective in patients with Class II or III BRAF alterations which account for a large proportion (34%) of BRAF mutations. KIN-2787 is an orally available, potent and selective small molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers, in addition to Class I mutants. Methods: The efficacy and tolerability of the pan-RAF inhibitor, KIN-2787, was evaluated in vitro and in vivo in Class I, II, and III BRAF mutation-driven human cancer models. Results: In biochemical assays, KIN-2787 showed low nanomolar to picomolar potency against RAF1, BRAF, and ARAF (IC50 0.06-3.46 nM) with minimal activity towards non-RAF kinases. In cell-based assays, KIN-2787 inhibited RAF activity, as measured by inhibition of downstream ERK phosphorylation (pERK), across multiple BRAF mutant cancer cell lines. Class II and III BRAF mutant cell lines were the most responsive when treated with KIN-2787 (IC50 &lt; 50 nM); 19- and 7-fold more sensitive compared to cells harboring wild-type BRAF, respectively. Dose-dependent inhibition of A-375 (Class I), BxPC-3 (Class II), and WM3629 (Class III) BRAF mutant human xenograft tumor growth was attained with daily KIN-2787 treatment and was well-tolerated. A trend towards greater tumor responses was observed with twice daily (BID) compared to once daily (QD) dosing of KIN-2787; however, the two dosing regimens led to similar tumor growth inhibition (TGI) and regressions (mean TGI up to 101-118%; p ≤0.0001) at equivalent total daily doses. Furthermore, KIN-2787 led to a significant in vivo pharmacodynamic response using either regimen, however, prolonged target coverage, as measured by pERK, was achieved with BID dosing. The impact of KIN-2787 treatment on additional biomarkers, including transcriptional changes and MAPK pathway modulation in cell-based models and patient-derived samples, will be presented at the meeting. Conclusions: KIN-2787 is a next-generation pan-RAF inhibitor with pronounced in vitro and in vivo activity against human cancers driven by Class II and III BRAF mutations. A phase 1 dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 monotherapy in patients with advanced or metastatic solid tumors harboring BRAF gene alterations, including Class II and III mutations, is expected to initiate in 2021.

FIREFLY-1: A phase 2 study of the pan-RAF inhibitor DAY101 in pediatric patients with low-grade glioma.

TPS10056 Background: The mitogen-activated protein kinase (MAPK) signaling pathway is an essential pathway that regulates key cell functions such as growth, survival, and differentiation. Genomic alterations and dysregulation of the MAPK pathway including BRAF fusions, point mutations (e.g. BRAF V600) and in-frame deletions have been described in many different types of malignancies, including pediatric low-grade glioma (pLGG) and other pediatric cancers. The identification of the KIAA1549:BRAF fusion in 2008 led to deeper understanding of the genomic events driving growth of pLGG (Jones, Cancer Res 2008). Despite the low-grade histology and excellent long-term survival, pLGGs are often associated with tumor- and treatment-associated morbidity and significant late-effects that persist throughout the lifespan of the patient. DAY101 is an oral, highly selective, CNS-penetrant small-molecule, Type II pan-RAF kinase inhibitor that is being developed for patients with pLGG harboring an activating BRAF-alteration. DAY101 has demonstrated tumor inhibition in preclinical models and has achieved clinically meaningful and durable responses in 7/8 patients with RAF-altered LGG in a pediatric phase 1 trial, including 2 complete responses, 3 partial responses, 2 stable disease and 1 progressive disease with a median time to response of 10.5 weeks. Patients have been treated for up to two years with no discontinuations due to toxicity or disease progression (Wright, SNO 2020). Methods: FIREFLY-1 is an open-label, multi-center, international Phase 2 study with DAY101 in pediatric and young adult patients between the ages of 6 months and 25 years with LGG harboring a documented BRAF-alteration as determined by local laboratory testing. DAY101 is administered orally once a week on a continuous 28-day schedule. Patients who respond will be treated for a minimum of two years after which they may at any point, opt to enter a “drug holiday” discontinuation period. Dosing is based on body surface area. DAY101 is available in a pediatric-friendly oral liquid formulation and tablets. The primary endpoint is objective response rate based on Response Assessment for Neuro-Oncology (RANO) as determined by an independent review committee. Secondary endpoints include objective response rate based on Response Assessment in Pediatric Neuro-Oncology (RAPNO) working group criteria, duration of response and safety. Exploratory endpoints include quality-of-life measurements as well as functional outcomes. Molecular abnormalities will be characterized through the analysis of archival tissue. Enrollment began in February 2021 and is ongoing. Clinical trial information: NCT03429804.

Patient-derived tumor xenograft and organoid models established from resected pancreatic, duodenal and biliary cancers

Patient-derived xenograft (PDX) and their xenograft-derived organoid (XDO) models that recapitulate the genotypic and phenotypic landscape of patient cancers could help to advance research and lead to improved clinical management. PDX models were established from 276 pancreato-duodenal and biliary cancer resections. Initial, passage 0 (P0) engraftment rates were 59% (118/199) for pancreatic, 86% (25/29) for duodenal, and 35% (17/48) for biliary ductal tumors. Pancreatic ductal adenocarcinoma (PDAC), had a P0 engraftment rate of 62% (105/169). KRAS mutant and wild-type PDAC models were molecularly profiled, and XDO models were generated to perform initial drug response evaluations. Subsets of PDAC PDX models showed global copy number variants and gene expression profiles that were retained with serial passaging, and they showed a spectrum of somatic mutations represented in patient tumors. PDAC XDO models were established, with a success rate of 71% (10/14). Pathway activation of KRAS-MAPK in PDXs was independent of KRAS mutational status. Four wild-type KRAS models were characterized by one with EGFR (L747-P753 del), two with BRAF alterations (N486_P490del or V600E), and one with triple negative KRAS/EGFR/BRAF. Model OCIP256, characterized by BRAF (N486-P490 del), had activated phospho-ERK. A combination treatment of a pan-RAF inhibitor (LY3009120) and a MEK inhibitor (trametinib) effectively suppressed phospho-ERK and inhibited growth of OCIP256 XDO and PDX models. PDAC/duodenal adenocarcinoma have high success rates forming PDX/organoid and retaining their phenotypic and genotypic features. These models may be effective tools to evaluate novel drug combination therapies.