RAF Dimerization Research Articles

MEK inhibitors lead to PDGFR pathway upregulation and sensitize tumors to RAF dimer inhibitors in NF1-deficient malignant peripheral nerve sheath tumor (MPNST).

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive subtype of soft tissue sarcoma with a high propensity to metastasize and extremely limited treatment options. Loss of the RAS-GAP NF1 leads to sustained RAF/MEK/ERK signaling in MPNST. However, single-agent MEK inhibitors (MEKi) have failed to elicit a sustained inhibition of the MAPK signaling pathway in MPNST. We employed pharmacological, biochemical, and genetic perturbations of the receptor tyrosine kinase (RTK) and MAPK signaling pathway regulators to investigate the mechanisms of MEKi resistance and evaluated combination therapeutic strategies in various preclinical MPNST models in vitro and in vivo. Here, we report that MEKi treatment resistance in MPNST involves two adaptive pathways: direct transcriptional upregulation of the receptor tyrosine kinase (RTK) PDGFRβ, and MEKi-induced increase in RAF dimer formation and activation of downstream signaling. While the pharmacological combination of MEKi with a PDGFRβ specific inhibitor was more effective than treatment with MEKi alone, the combination of MEKi and RAF-dimer inhibitors led to a robust inhibition of the MAPK pathway signaling. This combination treatment was effective in vitro and in vivo, as demonstrated by the significant increase in drug synergism and its high effectiveness in decreasing MPNST viability. Our findings suggest that the combination of MEKi and PDGFR and/or RAF dimer inhibitors can overcome MEKi resistance and may serve as a novel targeted therapeutic strategy for NF1-deficient MPNST patients, which in turn could impact future clinical investigations for this patient population.

A phase 1, open-label, dose escalation and dose expansion study to evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of PF-07799544 (ARRY-134) as a single agent and in combination with PF-07799933 BRAF dimer inhibitor, in participants 16 years and older with advanced solid tumors.

TPS3180 Background: The MAPK pathway plays a role in several key signaling and phosphorylation events that contribute to tumorigenesis. Inhibition of MEK, along with RAF kinases, has proven to be a successful transformative strategy for melanoma and other MAPK pathway-altered tumors. However, the duration of clinical benefit is limited by a narrow therapeutic index, de novo and acquired resistance and poor brain penetration. PF-07799544 is a next-generation, fully brain penetrant MEK inhibitor. PF-07799933 is an oral selective ATP-competitive small-molecule RAF kinase inhibitor that suppresses BRAF signaling in BRAF V600-mutant and non-V600-BRAF mutant tumors. It displays significantly less paradoxical activation than approved BRAFi and is not “pan RAF” as it spares non-BRAF mutated-containing RAF dimers. We describe here the design of the Phase 1 study of PF-07799544 as Monotherapy or in Combination with a next generation BRAF dimer inhibitor PF-07799933 in participants with BRAF mutated (BRAFm) advanced solid tumors. Methods: The purpose of this first-in-human study is to investigate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and potential clinical benefits of PF-07799544 administered as a single agent in participants with advanced solid tumors (Phase 1a) and in combination with PF-07799933 in participants with BRAF Class 1, 2 and 3 mutated solid tumors with or without brain involvement (Phase 1b). Phase 1a will enroll participants with advanced solid tumors who have progressed on standard of care. The primary objective is to determine monotherapy MTD/RDE of PF-07799544. Once PK measurements indicate the potential for significant WT MEK mutation target coverage, participants with untreated and symptomatic brain metastases (bm) will be allowed to enroll. Phase 1b is comprised of multiple sub-studies, all of which evaluate PF-07799544 in combination with PF-07799933 in participants with BRAFm solid tumors who have progressed on standard of care therapies, including Class 1 BRAFi where indicated. Sub-study B: BRAFm melanoma (V600 and non-V600) will determine the MTDc/RDEc of the combination (Part 1 primary objective), followed by dose expansion cohorts (Part 2) (~80 participants): Cohort 1: BRAF V600 melanoma (asymptomatic and untreated bm permitted), Cohort 2: BRAF V600 melanoma (symptomatic bm), Cohort 3 BRAFm Class 2/3 melanoma (asymptomatic and untreated bm permitted); Sub-study C: BRAFm Class 2 or 3 advanced solid tumor Cohort 1 (asymptomatic and untreated bm permitted and Cohort 2 (symptomatic bm) (~20 each cohort). The main objectives of the dose expansion cohorts in all substudies, will be to evaluate anti-tumor activity, safety, PK and PD. Clinical trial information: NCT05538130 .

Abstract 593: DCC-3084, a brain penetrant RAF dimer inhibitor, broadly inhibits BRAF class I, II, and III alterations leading to growth inhibition of intracranially implanted tumors in preclinical models

Abstract Background: Mutations in the RAS/MAPK pathway are a frequent driver of cancer, with oncogenic RAS or RAF mutations occurring in >30% of all cancers. First generation BRAF inhibitors are approved for use for tumors with Class I BRAF mutations (V600X). However, these drugs are not efficacious in RAF dimer mutant and RAS mutant cancers due to paradoxical activation of RAF dimers. Herein, we describe DCC-3084, a brain penetrant, potent and selective investigational switch-control inhibitor of BRAF and CRAF kinases, shown preclinically to target all relevant aberrant signaling mechanisms (monomers, heterodimers and homodimers) and inhibits growth of subcutaneously and intracranially implanted tumors in preclinical models. Methods: Inhibition of RAF kinases was measured using recombinant enzymes. Cellular proliferation was measured using resazurin to monitor cell viability. Inhibition of ERK or RSK phosphorylation was measured by AlphaLISA or ELISA. Pharmacokinetics (PK) in the plasma, brain and CSF compartments were measured following oral dosing in Wistar rats. Subcutaneous or intracranial BRAF mutant mouse xenograft models were used to assess PK, pharmacodynamics (PD), and efficacy. Results: DCC-3084 treatment resulted in potent single-agent inhibition of MAPK pathway signaling, and cellular proliferation in a wide range of Class I, II, III BRAF and BRAF fusion altered cell lines. DCC-3084 was demonstrated to be CNS penetrable and exhibited dose dependent oral exposure with robust inhibition of the MAPK pathway in PK/PD models. Oral treatment of DCC-3084 as a single agent resulted in tumor regression in subcutaneously implanted BRAF Class I and BRAF fusion xenograft models. Additionally, DCC-3084 resulted in tumor regression or growth inhibition in intracranially implanted BRAF Class I and BRAF fusion tumor models. Conclusions: The switch-control inhibitor DCC-3084 broadly inhibits Class I, II and III BRAF mutations, BRAF fusions, and BRAF/CRAF heterodimers leading to tumor regression in preclinical models. DCC-3084 CNS penetration enables potential for further research in brain metastases or primary brain cancer. Citation Format: Gada Al-Ani, Stacie L. Bulfer, Jefferey D. Zwicker, Chase K. Crawley, Kristin M. Elliot, Salim Javed, Qi Groer, Molly M. Hood, Joshua W. Large, Kylie Luther, Yeni K. Romero, Forrest A. Stanley, Daniel C. Tanner, Hanumaiah Telikepalli, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DCC-3084, a brain penetrant RAF dimer inhibitor, broadly inhibits BRAF class I, II, and III alterations leading to growth inhibition of intracranially implanted tumors in preclinical models [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 593.

Abstract 296: BRAFi-induced ROCK-mediated non-canonical nuclear β-catenin shuttling drives a phenotypic switch in cancer-associated fibroblasts

Abstract The discovery of BRAF mutations in around 60% of melanoma patients and the subsequent development of targeted inhibitors to counter the hyperactive BRAF-driven pro-tumorigenic signaling pathway have marked a significant milestone in melanoma therapy. However, despite this progress, the long-term outcomes remain unsatisfactory for many patients due to the occurrence of drug resistance. We previously reported that cancer-associated fibroblasts (CAFs) are stimulated by BRAF inhibitors (BRAFi) to drive matrix remodeling and therapeutic escape pathways in melanoma cells. However, the underlying mechanism by which CAFs are reprogrammed by BRAFi to obtain enhanced phenotypes remains elusive. Here, we present compelling evidence showing that BRAFi (PLX4032), but not CRAF inhibitor (GW5074) or pan-RAF inhibitor (RAF709), induces the accumulation of nuclear β-catenin in CAFs harboring wild-type BRAF, a process mediated by BRAF and CRAF but not ARAF isoform. BRAFi binding to BRAF and CRAF leads to robust RAF kinase dimerization, including BRAF and CRAF homodimer and BRAF: CRAF heterodimer. RAF dimers are subsequently recruited to the plasma membrane, where BRAF and CRAF are phosphorylated on residues Thr599 and Ser338, respectively, and activated in a RAS-GTP-dependent manner. RAF activation stimulates the downstream Rho kinase (ROCK) and MEK/ERK signaling pathways simultaneously. Notably, ablating RAS and RAF isoforms effectively suppressed BRAFi-induced activation of ROCK1/2 signaling in CAFs, further confirming that the ROCK pathway is a downstream effector of BRAFi-driven RAF activation and is intricately linked to increased nuclear β-catenin in CAFs. The pharmacological blockade of ROCK activity, using HA-1077, diminished BRAFi-induced nuclear β-catenin accumulation in CAFs, suggesting BRAFi-driven cytoskeletal remodeling plays a pivotal role in the nuclear shuttling of β-catenin. Using a mouse model that mimics human BRAF-mutant melanoma, we found that elevated level of nuclear β-catenin drives the conversion of fibroblasts into α-SMA-positive CAFs, leading to elevated collagen deposition and contributing to melanoma progression in vivo. In summary, BRAFi reprograms the transcriptional activity in CAFs by driving increased nuclear β-catenin to increase their ability to remodel the tumor ECM and promote melanoma cell proliferation. Collectively, the data offer new insights into the molecular mechanisms that underlie the paradoxical reprogramming of CAFs by BRAFi in melanoma therapy through the non-canonical β-catenin pathway. Citation Format: Bruna da Silva Soley, Radhika Athalye, Ryan Zhang, Thomas Andl, Yuhang Zhang. BRAFi-induced ROCK-mediated non-canonical nuclear β-catenin shuttling drives a phenotypic switch in cancer-associated fibroblasts [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 296.

Abstract 1972: Preclinical activity of the type II RAF inhibitor tovorafenib in tumor models harboring either a BRAF fusion or a NF1-LOF mutation

Abstract Genomic alterations and dysregulation of the MAPK pathway have been described in many different types of cancers. BRAF V600 point mutations and BRAF fusions, which are found in both pediatric and adult cancers, are oncogenic drivers that drive constitutive activation of the RAF pathway (Jones 2009, Yaeger 2019). Neurofibromatosis 1 (NF1) loss-of-function (LOF) mutations, which occur in many cancer types, result in decreased neurofibromin GAP function, thus activating RAS (Basu 1992). Tovorafenib is an investigational, selective, CNS-penetrant, small molecule type II RAF inhibitor which inhibits both RAF monomers and dimers and has been shown preclinically to impact the KIAA1549::BRAF fusion (Sun 2017). In this study, the impact of tovorafenib alone or in combination with MEK inhibitor, pimasertib, was explored in adult or pediatric tumor models harboring BRAF fusions or NF1-LOF mutations. At clinically relevant doses, tovorafenib treatment resulted in tumor regression in an AGK::BRAF fusion melanoma patient-derived xenograft (PDX) model in vivo. In contrast, little anti-tumor activity was observed in response to tovorafenib in vivo in two tumor models harboring NF1-LOF mutations. Ongoing PK-PD studies in both the AGK::BRAF fusion model and the NF1-LOF models will assess the extent and duration of target inhibition. In vitro, anti-proliferative activity of tovorafenib was also evaluated in NF1-LOF mutant tumor cell lines or PDX models of different tumor types, including melanoma, lung and malignant peripheral nerve sheath tumor (MPNST). While tovorafenib was potent in the BRAF V600E tumor cell line, little activity was observed in the NF1-LOF tumor cell lines. Modulation of phosphorylated-ERK (pERK) was also assessed. At very low concentrations, modest induction of pERK was observed in the NF1-LOF tumor cell lines, with inhibition at higher concentrations. In vitro combination studies were conducted using pimasertib and tovorafenib, or a tool compound. Synergy was observed in a NF1-LOF embryonal rhabdomyosarcoma PDX model ex vivo and a NF1-LOF MPNST cell line in vitro, suggesting that vertical pathway inhibition is impactful in the NF1-LOF mutant setting. Tovorafenib is currently being evaluated as a monotherapy in relapsed or progressive pediatric low-grade glioma (pLGG) harboring RAF alterations (NCT04775485) and in combination with pimasertib in patients ≥12 years of age with recurrent, progressive, or refractory solid tumors harboring MAPK pathway alterations (NCT04985604). Ongoing preclinical translational work will continue to explore potential biomarker-defined tumor indications for these agents. Citation Format: Shubhra Rastogi, Sam Perino, Madhu Lal-Nag, Yujin Wang, Samuel C. Blackman, Eleni Venetsanakos. Preclinical activity of the type II RAF inhibitor tovorafenib in tumor models harboring either a BRAF fusion or a NF1-LOF mutation [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 1972.

Abstract 598: HSK42360: A potent, brain permeable, BRAF paradox breaker for the treatment of BRAF-driven cancers

Abstract Mutations in the BRAF gene are among the most common identified in human cancers approximately 7% of all cancers evaluated, including 70% of melanomas, 50% of thyroid-like cancers, 10% to 20% of colorectal cancers, 13% of serous ovarian cancers, 3-7% of non-small-cell lung cancers, and most recently, hairy cell leukemia. Approved BRAF inhibitors (or +MEKi) against BRAFV600E/K improved initial clinical efficacy. However intrinsic and acquired resistance often arise through BRAF paradoxical activation, limiting durable responses to current BRAF inhibitors. In addition, Approved BRAFi responds poorly to patients with BRAF mutant gliomas or brain metastases, mainly due to limited brain permeability. More effective therapeutic options are therefore needed. To address this clinical challenge, we developed a potent, brain-penetrating paradox breaker HSK42360, as a promising therapy for patients with BRAFV600E mutation, progression or brain metastases following treatment with approved BRAFi or BRAFi/MEKi therapies. HSK42360 is a potent BRAFV600E inhibitor with IC50 value of 5 nM at enzymatic level. Further characterization showed that HSK42360 did not induce homodimer or heterodimer of CRAF with CRAF or BRAF and thereby blocked reactivation of the MAPK pathway in HCT116 cells, whereas Dabrafenib significantly induced RAF dimerization and MAPK reactivation. HSK42360 exhibited significant anti-proliferation activity against multiple tumor cell lines with BRAFV600E, including A375 (melanoma), DU4475 (breast cancer), and COLO205 (colon cancer), but was highly selective for BRAF wild-type cells. HSK42360 was administered orally once daily and showed broad-spectrum antitumor activity in the COLO205 and A375 CDX models. Immunohistochemistry showed a close correlation between the inhibition of tumor growth and ERK1/2 phosphorylation in A375 CDX tumor tissues. To explored the impact of HSK42360 in a brain micro-metastasis model aimed at mimicking early CNS metastatic spread, tumor progression was monitored through BLI (full name) imaging, which revealed that HSK42360 was highly brain penetrant with Kp,uu greater than 1 and triggered potent antitumor activity and prolonged survival in brain metastatic models of melanoma. We next tested the hypothesis that HSK42360 could retain activity in models presenting RAF dimer-driven resistance. Surprisingly, HSK42360 demonstrated promising antitumor effects in the first gen-resistant NRASmA375(NRASQ61K, BRAFV600E) CDX model, supporting its use as a follow-on therapy for current BRAFi resistance. Collectively, these preclinical studies validated that HSK42360 is a novel brain-permeable BRAF paradox breaker that exhibits outstanding antitumor activity in brain metastasis and RAF dimer-driven resistance. Citation Format: Shidong Gao, Yangguang Li, Ju Wang, Pingming Tang, Zongjun Shi, Yao Li, Pangke Yan. HSK42360: A potent, brain permeable, BRAF paradox breaker for the treatment of BRAF-driven cancers [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 598.

Abstract 6623: Ala59 mutants of KRAS cooperate with Nf1 loss to enhance colon tumorigenesis

Abstract Understanding the role of KRAS allele heterogeneity in tumor development, and its influence on therapeutic efficacy, is critical for the improvement of cancer treatment. The Kirsten rat sarcoma (KRAS) gene is the most frequently mutated oncogene in colorectal cancer (CRC) and patients with CRC tumors positive for KRAS mutations have poorer outcomes and limited treatment options. Extensive work over the past four decades has shown that common oncogenic alleles of KRAS are associated with hyperactivation of the MAPK signaling pathway (i.e. RAF/MEK/ERK), and it is for this reason that patients with KRAS mutant tumors are excluded from targeted therapies against EGFR. Recently, we characterized two alleles of KRAS, KRASA59T and KRASA59E, that paradoxically promote KRAS activation but are impaired in their ability to promote BRAF dimerization (Johnson et al. Mol Cell. 2022. PMID: 35202574). Consistent with these biochemical functions, KRASA59T and KRASA59E are enriched in CRC tumors that already have genetic alterations in genes that regulate MAPK signaling (e.g. EGFR, BRAF, NRAS, NF1). Further, unlike other described oncogenic alleles of KRAS, clinical data suggest that these KRASA59T and KRASA59E may not confer intrinsic resistance to anti-EGFR therapies at all. While KRASA59T and KRASA59E may not promote BRAF dimers, here we show that KRASA59T and KRASA59E does promote dimerization between ARAF and CRAF, and using an unpublished conditional mouse mode of K-Ras (i.e. LSL-K-RasA59E) we tested (1) whether Ala59 alleles are oncogenic or (2) require cooperation with additional MAPK pathway activating mutations, and (3) whether Ala59 alleles can promote intrinsic resistance to EGFR inhibition. Here, we show that endogenous K-RasA59E can disturb colon homeostasis and decrease mouse survival in an autochthonous colon tumor model, demonstrating that A59E alone is an oncogenic allele of KRAS in the colon. Breeding of LSL-K-RasA59E mice with mice harboring a conditional knockout of Nf1 (i.e. Nf1fl/fl), a GTPase activating protein (GAP) that promotes activation of wildtype RAS, show that combined expression of K-RasA59E and wildtype RAS activation cooperatively perturb colon homeostasis and decrease mouse survival. Thus, while KRASA59E may not require a second genetic alteration to activate the MAPK signaling pathway, it can cooperate with these alterations to increase the severity of disease. Finally, using mouse colon derived organoids, we show that endogenous K-RasA59E neither promotes EGF independence of organoid growth nor confers on these organoids a resistance to EGFR inhibition. Thus, our data support a unique role for RAF dimer function in in KRAS mutant tumors of the colon in both development and sensitivity to drugs, as well as argue for the inclusion of KRASA59T and KRASA59E bearing CRC tumors in anti-EGFR therapies. Citation Format: Christian William Johnson, Elizabeth M. Terrell, Josh Cook, Bing Shui, Eve O'Donoghue, Shannon Hull, Moon Hee Yang, Shikha Sheth, Rona Yaeger, Scott Kopetz, Deborah K. Morrison, Kevin Haigis. Ala59 mutants of KRAS cooperate with Nf1 loss to enhance colon tumorigenesis [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 6623.

Bioluminescence Resonance Energy Transfer (BRET)-based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

The CNK–HYP scaffolding complex promotes RAF activation by enhancing KSR–MEK interaction

The RAS–MAPK pathway regulates cell proliferation, differentiation and survival, and its dysregulation is associated with cancer development. The pathway minimally comprises the small GTPase RAS and the kinases RAF, MEK and ERK. Activation of RAF by RAS is notoriously intricate and remains only partially understood. There are three RAF isoforms in mammals (ARAF, BRAF and CRAF) and two related pseudokinases (KSR1 and KSR2). RAS-mediated activation of RAF depends on an allosteric mechanism driven by the dimerization of its kinase domain. Recent work on human RAFs showed that MEK binding to KSR1 promotes KSR1–BRAF heterodimerization, which leads to the phosphorylation of free MEK molecules by BRAF. Similar findings were made with the single Drosophila RAF homolog. Here we show that the fly scaffold proteins CNK and HYP stabilize the KSR–MEK interaction, which in turn enhances RAF–KSR heterodimerization and RAF activation. The cryogenic electron microscopy structure of the minimal KSR–MEK–CNK–HYP complex reveals a ring-like arrangement of the CNK–HYP complex allowing CNK to simultaneously engage KSR and MEK, thus stabilizing the binary interaction. Together, these results illuminate how CNK contributes to RAF activation by stimulating the allosteric function of KSR and highlight the diversity of mechanisms impacting RAF dimerization as well as the regulatory potential of the KSR–MEK interaction.

Cross-Species Comparison of the Pan-RAF Inhibitor LY3009120's Anti-Tumor Effects in Equine, Canine, and Human Malignant Melanoma Cell Lines.

Malignant melanomas (MMs) are the abnormal proliferation of melanocytes and are one of the lethal skin cancers in humans, equines, and canines. Accordingly, MMs in companion animals can serve as naturally occurring animal models, completing conventional cancer models. The common constitutive activation of the MAPK and PI3K pathways in MMs has been described in all three species. Targeting the related pathways is considered a potential option in comparative oncologic approaches. Herein, we present a cross-species comparative analysis exposing a set of ten melanoma cell lines (one human, three equine, and six canine) derived from primary tumors or metastasis to a pan-RAF and RAF dimer inhibitor (LY3009120). Cellular response (proliferation, biomass, metabolism, early and late apoptosis/necrosis, and morphology) and the presence of pathogenic single-nucleotide variants (SNVs) within the mutational hotspot genes BRAF exon 11 and 15, NRAS exon 2 and 3, KRAS exon 2, and KIT exon 11 were analyzed. This study showed that equine malignant melanoma (EMM) cells (MelDuWi) harbor the KRAS p.Q61H mutation, while canine malignant melanoma (CMM) cells (cRGO1 and cRGO1.2) carry NRAS p.G13R. Except for EMM metastasis cells eRGO6 (wild type of the above-mentioned hotspot genes), all melanoma cell lines exhibited a decrease in dose dependence after 48 and 72 h of exposure to LY3009120, independent of the mutation hotspot landscape. Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in all melanoma cell lines except for eRGO6. The anti-tumor effects of LY3009120 were observed in nine melanoma cell lines, indicating the potential feasibility of experimental trials with LY3009120. The present study reveals that the irradiation-resistant canine metastasis cells (cRGO1.2) harboring the NRAS p.G13R mutation are significantly LY3009120-sensitive, while the equine metastases-derived eRGO6 cells show significant resistance to LY3009120, which make them both valuable tools for studying resistance mechanisms in comparative oncology.

Abstract A012: Emergence of MEK1 mutations in RAF or RAS-mutant solid tumors following treatment with a combination of selective type II RAF inhibitor belvarafenib and MEK inhibitor cobimetinib

Abstract Acquisition of ARAF mutations upon treatment with the potent and selective RAF dimer (type II) inhibitor belvarafenib has been previously observed, but whether these alterations or others are observed in patients treated with a combination of belvarafenib and the MEK inhibitor cobimetinib is unknown. The mutational profiles from circulating tumor DNA (ctDNA) were determined using FoundationOne liquid ctDNA assay from paired baseline (BL) and end of treatment (EOT) peripheral blood samples from 79 patients with locally advanced or metastatic RAF or RAS-mutant solid tumors treated with belvarafenib and cobimetinib in the HM-RAFI-103 trial (NCT03284502). The proportion of mutations were compared between BL and EOT using a McNemar’s test (unadjusted) to identify mutations significantly enriched or lost following treatment. ARAF mutations were not observed to significantly increase following treatment with belvarafenib + cobimetinib (4% BL vs 4% EOT in all solid tumors, p = 1; 6% BL vs 6% EOT in melanoma), and in patients with pre-existing ARAF mutations, the variant allele frequency did not consistently increase following treatment. The MEK1 gene had the greatest increase in mutations following treatment, present in 5% (4/79) patients at BL vs 16% (13/79) at EOT (p = 0.02) across all solid tumors, which included 44 colorectal cancer (CRC) cases (56%), and 18 melanoma (23%). While CRC comprised the largest fraction of all solid tumors in this study, it did not contribute the greatest gain in MEK1 mutations at EOT, with 7% (3/44) of CRC patients having MEK1 mutations at BL vs 14% (6/44) at EOT (p = 0.2). The indication with the greatest number of patients developing previously undetected MEK1 mutations at EOT was melanoma, where MEK1 mutations were present in 0% (0/18) patients at BL vs 28% (5/18) at EOT (p = 0.07). The MEK1 mutations gained in melanoma, which included Q58_E62del, P124L, and C121S, occurred in the N-terminal or catalytic core domains of MEK1, were known to be of functional impact, and all but one patient developing them had ≥ 1 known to be gain of function via the Clinical Knowledge Database; one patient gained three distinct MEK1 mutations following treatment. The MEK1 C121S mutation was previously observed to appear in a melanoma patient who developed resistance to the BRAF V600 inhibitor vemurafenib, and in vitro studies demonstrated it also conferred resistance to MEK inhibition (Wagle et al., 2011). Melanoma patients in the HM-RAFI-103 trial gaining MEK1 mutations at EOT were more commonly BRAF-mutant(mt) or NRAS non-mt melanoma at BL, but sample size is small and results were non-significant (p = 0.3 and 0.3, for association with BRAFmt and NRASmt, respectively, Fisher’s exact test). All melanoma patients who developed MEK1 mutations post treatment had either a partial response (1/5) or stable disease (4/5) as best overall response. Together, these data suggest the acquisition of gain of function MEK1 mutations following treatment of solid tumors, including melanoma, with combined type II RAF dimer and MEK inhibition. Citation Format: Stephanie Hilz, Marissa Chen, Malgorzata Nowicka, Harini Chakravarthy, Maryam Moshref, Luca Gerosa, Jennifer Eng-Wong, Cassie Chou, Young S. Noh, Yoon-hee Hong, Yibing Yan. Emergence of MEK1 mutations in RAF or RAS-mutant solid tumors following treatment with a combination of selective type II RAF inhibitor belvarafenib and MEK inhibitor cobimetinib [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A012.

The Discovery of Exarafenib (KIN-2787): Overcoming the Challenges of Pan-RAF Kinase Inhibition.

RAF, a core signaling component of the MAPK kinase cascade, is often mutated in various cancers, including melanoma, lung, and colorectal cancers. The approved inhibitors were focused on targeting the BRAFV600E mutation that results in constitutive activation of kinase signaling through the monomeric protein (Class I). However, these inhibitors also paradoxically activate kinase signaling of RAF dimers, resulting in increased MAPK signaling in normal tissues. Recently, significant attention has turned to targeting RAF alterations that activate dimeric signaling (class II and III BRAF and NRAS). However, the discovery of a potent and selective inhibitor with biopharmaceutical properties suitable to sustain robust target inhibition in the clinical setting has proven challenging. Herein, we report the discovery of exarafenib (15), a highly potent and selective inhibitor that intercepts the RAF protein in the dimer compatible αC-helix-IN conformation and demonstrates anti-tumor efficacy in preclinical models with BRAF class I, II, and III and NRAS alterations.

Abstract A090: Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations

Abstract Alterations in the RAS-MAPK pathway, such as mutations in NF1, RAS, and BRAF, often lead to oncogenic signaling and result in aberrant cell proliferation and tumor growth. Oncogenic BRAF mutations may be activated as either monomers or dimers, while oncogenic RAS mutations or loss-of-function mutations in NF1, a negative regulator of RAS, drive activation of RAF dimers to transduce MAPK signaling. Currently approved BRAF inhibitors are selective against monomeric BRAF mutations and largely inactive against dimeric RAF mutants. Additionally, these agents may also promote deleterious paradoxical activation of RAF in select contexts. Moreover, FDA-approved KRAS G12C mutant-selective inhibitors are ineffective for cancer patients who harbor other (non-G12C) KRAS, NRAS, and NF1 mutations which all lead to active RAF dimer. There remains a high unmet clinical need for a highly CNS penetrant RAF inhibitor that targets a broad spectrum of oncogenic RAF conformations in the context of RAF, RAS, and other upstream driver mutations. BDTX-4933 is a next generation MasterKey RAF inhibitor designed to address the shortcomings of previous generation RAF inhibitors. BDTX-4933 is an orthosteric inhibitor that potently targets the oncogenic RAF conformation shared by groups of RAF driver mutations and induces an inactive conformation of RAF. As a MasterKey inhibitor, BDTX-4933 potently inhibits a broad spectrum of BRAF mutations and active RAF dimers promoted by upstream oncogenic MAPK pathway alterations, such as KRAS, NRAS, and NF1 mutations. In vivo, once daily oral dosing of BDTX-4933 resulted in robust and sustained target engagement, inhibiting ERK phosphorylation without inducing paradoxical activation. In a mouse breadth of efficacy study with patient derived xenografts (PDX), BDTX-4933 demonstrated tumor growth inhibition and regression as a single agent across models expressing a variety of targeted BRAF or MAPK pathway oncogenic mutations. Additionally, BDTX-4933 as a single agent and in combination with a MEK inhibitor, demonstrates anti-tumor efficacy in KRAS mutant lung PDX models. In a PDX model of BRAF V600E positive tumor that progressed following BRAF and MEK combination therapy, BDTX-4933 achieved robust tumor growth regression. Furthermore, BDTX-4933 exhibits high CNS exposure leading to dose-dependent tumor growth inhibition, and survival benefit in mice implanted intracranially with xenograft BRAF mutant tumors. BDTX-4933 has a best-in-class RAF inhibitor profile to treat cancer patients harboring BRAF mutations or RAF dimer-promoting upstream genetic alterations. BDTX-4933 is currently in a Phase I clinical study in patients with solid tumor harboring sensitive BRAF and RAS alterations (NCT05786924). For more information on this active Phase 1, please contact: ClinicalTrials@bdtx.com. Citation Format: Yoon-Chi Han, Pui Yee Ng, Luisa Shin Ogawa, Shao Ning Yang, Miao Chen, Darlene Romashko, Tai-An Lin, Elizabeth Buck. Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A090.

Abstract LB_B10: Non-V600 BRAF mutants and their precise targeting

Abstract Hyperactive RAS/RAF/MEK/ERK signalling is responsible for a large portion of cancers and targeting this signalling pathway has achieved a promising outcome in clinic cancer treatment. However, significant challenges such as drug resistance are still existing. To resolve these problems, we need a deeper understanding of the regulatory mechanism and hence develop better targeting approaches against this signalling pathway. In this study, we investigated the molecular mechanism that controls that activity of BRAF, a key component of this signalling pathway, and found that BRAF cycled between inactive monomeric status and active dimeric status, which is coordinatively regulated by Cdc37/Hsp90 chaperone and 14-3-3 scaffold. Further, we found that most non-V600 mutations of BRAF in cancer genomes undermined this regulatory mechanism through disturbing the association of BRAF with Cdc37/Hsp90 chaperone or 14-3-3 scaffold and hence trapped BRAF in active dimeric status. Given the high dimer affinity of these non-V600 BRAF mutants, we next examined whether their activity could be inhibited effectively RAF dimer breaker PLX8394 in vitro and in vivo. Indeed, we found that in contrast to clinic RAF inhibitor Vemurafenib (PLX4720), PLX8394 strongly inhibited the activity of non-V600 BRAF mutants and induced a rapid regression of fibroblastomas driven by non-V600 BRAF mutants. Together, our data clearly illustrates how oncogenic non-V600 mutations break down the regulation of BRAF by Cdc37/Hsp90 chaperone and 14-3-3 scaffold, and how these BRAF mutations could be precisely targeted by RAF dimer breaker, which has important implications for anti-BRAF cancer therapy. Citation Format: Jiancheng Hu. Non-V600 BRAF mutants and their precise targeting [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B10.

Efficacy of Belvarafenib with and without Cobimetinib in Preclinical Models of Ras Pathway-Mutant AML

Introduction Outcomes for children with acute myeloid leukemia (AML) remain poor, with ~40% dying from refractory leukemia or treatment-related toxicity (Gamis et al. 2014). NRAS, KRAS, and NF1 mutations occur in over 40% of pediatric AMLs (Bolouri et al. 2018), but efforts to therapeutically target the RAS/mitogen activated protein kinase (MAPK) pathway have been largely unsuccessful. Belvarafenib, a novel type II pan-RAF kinase inhibitor that inhibits mutant monomeric BRAF proteins and activated RAF homo- and heterodimers, has shown promising safety/efficacy data in adult solid cancers (Yen et al. 2021). Here, we investigate belvarafenib in preclinical models of AML, both alone and in combination with the allosteric MEK inhibitor, cobimetinib. Methods We used a panel of NRAS (OCI-AML3, HL-60, THP-1) or KRAS (NOMO-1, NB4, and SKM-1) mutant human AML cell lines. Viability was determined by CellTiter-Glo. Synergy was assessed by Bliss Independence and Chou Talalay methods. Transcriptome and proteomic profiling were performed as previously described (Pucciarelli et al. 2020). Mouse AMLs were generated using retroviral insertional mutagenesis (Li et al. 2011). Cryopreserved primary AML cells were injected intravenously into sublethally irradiated recipients that were then treated daily with vehicle, belvarafenib, cobimetinib, or the combination until disease progression. Survival curves were generated using Kaplan-Meier analysis. Results Belvarafenib inhibited the growth of AML cells lines with nanomolar potency at IC50 values ranging from 48nM (OCI-AML3) to 310nM (SKM-1). In all cell lines, belvarafenib and cobimetinib were highly synergistic. Western blotting of OCI-AML3 cells treated with either belvarafenib or cobimetinib at their respective IC50 values (50nM belvarafenib, 20nM cobimetinib) for 4 or 24 hours demonstrated discordant effects on downstream MAPK effector proteins (Fig. 1). Whereas cobimetinib potently suppressed phosphorylated ERK (pERK), belvarafenib unexpectedly had no effect. Phosphorylated S6 (pS6) levels were not reduced by either belvarafenib or cobimetinib at their respective IC50 values, but were dramatically reduced by the drug combination. To further investigate these unanticipated biochemical findings, we treated OCI-AML3 cells with belvarafenib, cobimetinib or the combination and performed kinome profiling and transcriptome sequencing (RNAseq) analysis. Exposure to 10nM cobimetinib inhibited several kinases at 4 hours with rebound activation of kinases involved in cell cycle progression at 24 hours. Cells treated with 100nM belvarafenib displayed down-regulation of multiple kinases that persisted at 24 hours. Cells treated with the combination at these doses exhibited the most potent inhibition of many kinases; this effect recapitulated the effect of high dose (500nM) belvarafenib, but not high dose (50nM) cobimetinib. At the transcriptional level, cells treated with the combination showed far greater suppression of negative regulators of MAPK signaling (e.g., DUSPs, SPRY2/4, SPRED1/2) than either agent alone, suggesting potent inhibition of the MAPK pathway by the combination. Intriguingly, OCI-AML3 cells treated with high-dose belvarafenib or low-dose combination unexpectedly exhibited profound down-regulation of mTORC1-regulated genes on GSEA analysis. We extended these in vitro data by performing preclinical trials in mice transplanted with 5 independent primary Nras- or Kras-mutant AMLs that received belvarafenib (15mg/kg), cobimetinib (2mg/kg), the combination or vehicle by oral gavage (Fig. 2). The combination was well tolerated. Belvarafenib prolonged survival in all 5 trials; in 3 of 5, the addition of low-dose cobimetinib further enhanced survival (median survival: vehicle, 9 days; cobimetinib, 13 days; belvarafenib, 22 days; combination, 32 days; p < 0.0001). Conclusions Belvarafenib showed activity in 6 of 6 Ras mutant human AML cell lines and in 5 of 5 primary Ras mutant murine AMLs. Belvarafenib and cobimetinib displayed synergy in all AML cell lines and in 3 of 5 murine AMLs treated in vivo. Mechanistically, we identified distinct biochemical and transcriptional effects of RAF dimer and MEK inhibition in AML cells. We are characterizing these further and pursuing causes of resistance in primary Nras- and Kras-mutant mouse AMLs that relapsed after an initial response to treatment.

Protomer selectivity of type II RAF inhibitors within the RAS/RAF complex

RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement, but not engagement of BRAF or CRAF, is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and reveal poor ARAF protomer vulnerability for a cohort of RAF inhibitors undergoing clinical evaluation.

A novel phosphorylation site involved in dissociating RAF kinase from the scaffolding protein 14-3-3 and disrupting RAF dimerization

Rapidly accelerated fibrosarcoma (ARAF, BRAF, CRAF) kinase is central to the MAPK pathway (RAS-RAF-MEK-ERK). Inactive RAF kinase is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane via RAS-GTP, leading to the relief of autoinhibition, phosphorylation of key regulatory sites, and dimerization of RAF protomers. Although it is well known that active and inactive BRAF have differential phosphorylation sites that play a crucial role in regulating BRAF, key details are still missing. In this study, we report the characterization of a novel phosphorylation site, BRAFS732 (equivalent in CRAFS624), located in proximity to the C-terminus binding motif for the 14-3-3 scaffolding protein. At the C terminus, 14-3-3 binds to BRAFpS729 (CRAFpS621) and enhances RAF dimerization. We conducted mutational analysis of BRAFS732A/E and CRAFS624A/E and revealed that the phosphomimetic S→E mutant decreases 14-3-3 association and RAF dimerization. In normal cell signaling, dimerized RAF phosphorylates MEK1/2, which is observed in the phospho-deficient S→A mutant. Our results suggest that phosphorylation and dephosphorylation of this site fine-tune the association of 14-3-3 and RAF dimerization, ultimately impacting MEK phosphorylation. We further characterized the BRAF homodimer and BRAF:CRAF heterodimer and identified a correlation between phosphorylation of this site with drug sensitivity. Our work reveals a novel negative regulatory role for phosphorylation of BRAFS732 and CRAFS624 in decreasing 14-3-3 association, dimerization, and MEK phosphorylation. These findings provide insight into the regulation of the MAPK pathway and may have implications for cancers driven by mutations in the pathway.

Analysis of RAS and drug induced homo- and heterodimerization of RAF and KSR1 proteins in living cells using split Nanoluc luciferase

The dimerization of RAF kinases represents a key event in their activation cycle and in RAS/ERK pathway activation. Genetic, biochemical and structural approaches provided key insights into this process defining RAF signaling output and the clinical efficacy of RAF inhibitors (RAFi). However, methods reporting the dynamics of RAF dimerization in living cells and in real time are still in their infancy. Recently, split luciferase systems have been developed for the detection of protein–protein-interactions (PPIs), incl. proof-of-concept studies demonstrating the heterodimerization of the BRAF and RAF1 isoforms. Due to their small size, the Nanoluc luciferase moieties LgBiT and SmBiT, which reconstitute a light emitting holoenzyme upon fusion partner promoted interaction, appear as well-suited to study RAF dimerization. Here, we provide an extensive analysis of the suitability of the Nanoluc system to study the homo- and heterodimerization of BRAF, RAF1 and the related KSR1 pseudokinase. We show that KRASG12V promotes the homo- and heterodimerization of BRAF, while considerable KSR1 homo- and KSR1/BRAF heterodimerization already occurs in the absence of this active GTPase and requires a salt bridge between the CC-SAM domain of KSR1 and the BRAF-specific region. We demonstrate that loss-of-function mutations impairing key steps of the RAF activation cycle can be used as calibrators to gauge the dynamics of heterodimerization. This approach identified the RAS-binding domains and the C-terminal 14–3-3 binding motifs as particularly critical for the reconstitution of RAF mediated LgBiT/SmBiT reconstitution, while the dimer interface was less important for dimerization but essential for downstream signaling. We show for the first time that BRAFV600E, the most common BRAF oncoprotein whose dimerization status is controversially portrayed in the literature, forms homodimers in living cells more efficiently than its wildtype counterpart. Of note, Nanoluc activity reconstituted by BRAFV600E homodimers is highly sensitive to the paradox-breaking RAFi PLX8394, indicating a dynamic and specific PPI. We report the effects of eleven ERK pathway inhibitors on RAF dimerization, incl. third-generation compounds that are less-defined in terms of their dimer promoting abilities. We identify Naporafenib as a potent and long-lasting dimerizer and show that the split Nanoluc approach discriminates between type I, I1/2 and II RAFi.Ah1kwifp3aoZnF_eXdbERzVideo

Discovery of dual inhibitors of pan‐RAF and VEGFR2

AbstractColorectal cancer is among the most common and lethal malignancies globally, with K‐Ras mutations found in about 45% of colorectal cancer patients. Early‐stage BRAF‐specific inhibitors have been developed to prevent aberrant activation of RAS/RAF/MEK/ERK signaling caused by K‐Ras mutation, however, these BRAF‐specific inhibitors lead to RAF dimer formation and paradoxical CRAF activation. Consequently, there is a need to develop pan‐RAF inhibitors. In addition, it was reported that the inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) is crucial for the treatment of colorectal cancer. In this study, we designed and synthesized 94 N‐(phenyl)‐3‐(9H‐purin‐6‐yl)pyridine‐2‐amine derivatives, and discovered that N‐(5‐(3‐(9H‐purin‐6‐yl)pyridin‐2‐ylamino)‐2‐fluorophenyl)‐3‐(trifluoromethyl)benzamide (15h), 3‐(2‐cyanopropan‐2‐yl)benzamide derivative 16h, and 3,5‐bis(trifluoromethyl) benzamide derivative 17ab are the most potent dual inhibitors against LS513 (GI50 = 0.08, 0.2, and 0.3 μM, respectively) and VEGFR2 (IC50 = 0.01, 0.004, and 0.01 μM, respectively). These compounds are excellent preclinical candidates for the treatment of K‐Ras mutated colorectal cancer.

A phase 1, open-label, dose escalation and dose expansion study to evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of PF-07799933 (ARRY-440) as a single agent and in combination therapy in participants 16 years and older with advanced solid tumors with BRAF alterations.

TPS3164 Background: Approved BRAF inhibitors (BRAFi) are inactive against RAF dimers. Accordingly, the acquisition of BRAF dimers ( e.g., through drug-acquired splice variants or BRAF amplification) may lead to therapy resistance. Non-V600 (class II and III) BRAF alterations that occur de novo in diverse tumor types also signal as dimers and therefore have no approved targeted therapeutic options. Additional liabilities of approved BRAFi include toxicity from paradoxical activation of wild-type (WT) RAF and limited brain penetration, a common primary and metastatic site for BRAF-altered cancers. In contrast to approved agents, investigational BRAFi inhibit WT and mutant RAF proteins (A, B, and C-RAF, monomers, dimers), and therefore may be limited by on-target toxicity from “pan-RAF” inhibition. PF-07799933 is an oral selective ATP-competitive small-molecule RAF kinase inhibitor that suppresses BRAF signaling in BRAF V600-mutant and non-V600- BRAF mutant tumors. It displays significantly less paradoxical activation than approved BRAFi and is not “pan RAF” as it spares non-BRAF-containing RAF dimers. We describe here the Phase 1 study of PF-07799933 as monotherapy or in combination with binimetinib or cetuximab in participants with BRAF-altered advanced solid tumors. Methods: The purpose of this first-in-human study is to investigate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and potential clinical benefits of PF-07799933 administered as a single agent and in combination in participants with BRAF Class I, II and III mutated solid tumors with or without brain involvement. Part 1 (monotherapy dose escalation) and Part 2 (combination dose escalation) will enroll participants with BRAF V600 mutation who have progressed on a approved BRAFi or with de novo Class II or III alterations who have progressed on standard of care therapy. The primary objective of Part 1 is to determine monotherapy MTD/RDE of PF-07799933. Participants in Part 1 will be allowed to add on rational combination with either binimetinib or cetuximab at the time of disease progression. Once PK measurements indicate the potential for significant BRAF inhibition in the brain, participants with untreated and symptomatic brain metastases will be allowed to enroll. The primary objective of Part 2 is combination MTDc/RDEc of PF-07799933 with binimetinib or cetuximab. The primary objective of Part 3 is efficacy in defined cohorts (total ~120 participants): Cohort 1-2: BRAF V600 mutant melanoma; Cohort 3: BRAF Class II altered melanoma; Cohorts 4-5: BRAF V600 and Class II altered CRC; Cohort 6: other BRAF V600, Class II/III altered solid tumor not qualifying for Cohorts 1-5. Clinical trial information: NCT05355701 .