RAF Inhibitors Research Articles

RAS/RAF/MEK mutated high-grade serous ovarian cancer susceptible to Raf inhibition in vitro but not in vivo

RAS/RAF/MEK mutated high-grade serous ovarian cancer susceptible to Raf inhibition in vitro but not in vivo

Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity. Existing droplet-based microfluidic methods produce many cell-free droplets, underutilizing bead barcodes and reagents. Combinatorial indexing on microplates is more efficient for barcoding but labor-intensive. Here we present Overloading And unpacKing (OAK), which uses a droplet-based barcoding system for initial compartmentalization followed by a second aliquoting round to achieve combinatorial indexing. We demonstrate OAK’s versatility with single-cell RNA sequencing as well as paired single-nucleus RNA sequencing and accessible chromatin profiling. We further showcase OAK’s performance on complex samples, including differentiated bronchial epithelial cells and primary retinal tissue. Finally, we examine transcriptomic responses of over 400,000 melanoma cells to a RAF inhibitor, belvarafenib, discovering a rare resistant cell population (0.12%). OAK’s ultra-high throughput, broad compatibility, high sensitivity, and simplified procedures make it a powerful tool for large-scale molecular analysis, even for rare cells.

Novel therapies for pediatric low grade glioma.

Current biological findings provide new insights into the genetics driving growth of low-grade gliomas in pediatric patients. This has provided new targets for novel therapies. The purpose of this paper is to review novel therapies for pediatric low-grade gliomas that have been published in the past 24 months. Low-grade gliomas are often driven by mitogen activated protein kinase (MAPK) alterations either with BRAF V600E point mutations or BRAF fusions. Current advances have also highlighted novel fusions of fibroblast growth factor receptor (FGFR), myeloblastosis family of transcription factors (MYB), meningioma 1 tumor suppressor (MN1), neurotrophic receptor kinase family of receptors (NTRK), Kristen RAS (Rat Sarcoma Virus) oncogene homolog in mammals (KRAS), Receptor tyrosine kinase ROS proto oncogene 1 (ROS1), protein kinase C alpha (PRKCA), and platelet derive growth factor receptor (PDGFR) amplification. Novel therapies have been employed and are showing encouraging results in pediatric low-grade gliomas. Current trials are underway with newer generation pan RAF inhibitors and mitogen activated protein kinase - kinase (MEK) inhibitors. Other early phase clinical trials have provided safety data in pediatric patients targeting FGFR fusion, NTRK fusion, PDGFR amplification and ROS1 mutations. Historical treatment options in pediatric low-grade gliomas have utilized surgery, radiation therapy and conventional chemotherapy. Recently greater insight into their biology has found that alterations in MAPK driven pathways are often the hallmark of tumorigenesis. Targeting these novel pathways has led to tumor control and shrinkage without the use of conventional chemotherapy. Caution should be taken however, since these treatment options are still novel, and we do not fully appreciate the long-term effects. Nonetheless a new era of targeted medicine is here.

Preclinical Evaluation of a Radiolabeled Pan-RAF Inhibitor for RAF-Specific PET/CT Imaging.

Abnormalities in the RAS-RAF signaling pathway occur in many solid tumors, leading to aberrant tumor proliferation, invasion, and metastasis. Due to the elusive pharmacology of RAS, RAF inhibitors have become the main targeted therapeutic drugs. Naporafenib (LXH-254) is a high-affinity pan-RAF inhibitor with FDA Fast Track Qualification. We sought to develop an 18F-labeled molecular probe from LXH-254 for PET imaging of tumors overexpressing RAF to noninvasively screen patients for susceptibility to targeted RAF therapy. To reduce the lipid solubility, LXH-254 was designed with triethylene glycol di(p-toluenesulfonate) (TsO-PEG3-OTs) to obtain the precursor (LXH-254-OTs) and a nucleophilic substitution reaction with 18F to obtain the tracer ([18F]F-LXH-254). [18F]F-LXH-254 exhibited good molar activity (7.16 ± 0.81 GBq/μmol), radiochemical purity (>95%), and stability. Micro-PET imaging revealed distinct radioactivity accumulation of [18F]F-LXH-254 in tumors in the imaging groups, whereas in the blocked group, the tumor radioactivity level was consistent with the background tissue, illustrating the affinity and specificity of [18F]F-LXH-254 in targeting RAF. Overall, [18F]F-LXH-254 is a promising radiotracer for screening and diagnosing patients with RAF-related disease and monitoring their treatment. This is the first attempt at using an 18F-labeled RAF-specific radiotracer.

Abstract C011: Targeting STAT3 overcomes PDAC resistance to EGFR/RAF1 inhibition leading to complete and durable tumor regression

Abstract Pancreatic ductal adenocarcinoma (PDAC) belongs to the tumors with worst prognosis due to the lack of effective treatment. It was previously demonstrated that combined elimination of EGFR and RAF1, two main mediators of the KRAS signaling, induces complete regression of a limited subset of small tumors in a genetically engineered mouse model (Blasco et al., 2019). In the current study, we show that resistance of PDACs with big sizes to the elimination of RAF1 and EGFR is mediated by STAT3. Indeed, combined genetic ablation of EGFR, RAF1 and STAT3 both in vitro and in vivo, results in complete and irreversible regression of more than 40 independent mouse PDAC tumors driven by Kras/Trp53 mutations. Importantly, all PDACs were characterized by increased initial tumor size and highly heterogeneous molecular profile similar to that of Basal human tumors. Moreover, we have validated the efficacy of this novel therapeutic strategy using pharmacological agents, such as EGFR inhibitors approved for clinical use and the STAT3 PROTAC degrader SD-36. Finally, inhibition of RAF1, EGFR and STAT3 expression effectively blocked in vitro and in vivo tumor progression in ten independent patient-derived organoid (PDO) and patient-derived xenograft (PDX) cultures carrying KRAS and TP53 mutations. These results open the door to the development of novel targeted therapies for PDAC patients. Citation Format: Vasiliki Liaki, Mariano Barbacid, Carmen Guerra. Targeting STAT3 overcomes PDAC resistance to EGFR/RAF1 inhibition leading to complete and durable tumor regression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C011.

614MO Type II RAF inhibitor tovorafenib in recurrent/refractory (R/R) melanoma or other solid tumors with RAF fusions and/or RAF1 amplification

614MO Type II RAF inhibitor tovorafenib in recurrent/refractory (R/R) melanoma or other solid tumors with RAF fusions and/or RAF1 amplification

Genomic landscape of head and neck cancer in Asia: A comprehensive meta-analysis of 1016 samples

Head and neck cancer (HNC) is a diverse group of malignancies arising in the mucosal linings of the oral cavity, pharynx, and larynx, influenced by factors such as tobacco use, alcohol consumption, and human papillomavirus (HPV) infection. This study conducts a comprehensive meta-analysis of the mutational landscape of HNC across Asian cohorts, encompassing India, Korea, Japan, China, Singapore, and Saudi Arabia. The analysis highlights distinct genetic profiles influenced by environmental exposures, lifestyle habits, and genetic predispositions. Notably, the RAF family proteins, enriched in both Indian and Chinese cohorts, present potential therapeutic targets for RAF inhibitors like Vemurafenib. Additionaly, specific mutations like MET in Singaporean patients can be effectively addressed with drugs like Crizotinib, leading to rapid responses in HNSCC. Smokers exhibited high frequencies of CASP8 and FAT1 mutations. Novel driver genes, including RYR2 and ANK2, emerged with significant mutational frequencies in smokers. The RAS signaling pathway was identified as a prominent driver in HNC, contrasting with the globally prevalent PIK3CA/MTOR pathway. This study also underscores the high prevalence of HRAS mutations in Indian and Saudi cohorts. The study emphasizes the necessity for region-specific data to understand the unique molecular differences and develop effective therapies. The identification of NBEA and ANK2 as potential novel driver genes in HNC highlights new avenues for research and targeted therapeutic interventions tailored to the genetic profiles of Asian HNC patients.

Bromodomain-containing protein 4 knockdown promotes neuronal ferroptosis in a mouse model of subarachnoid hemorrhage.

Neuronal cell death is a common outcome of multiple pathophysiological processes and a key factor in neurological dysfunction after subarachnoid hemorrhage. Neuronal ferroptosis in particular plays an important role in early brain injury. Bromodomain-containing protein 4, a member of the bromo and extraterminal domain family of proteins, participated in multiple cell death pathways, but the mechanisms by which it regulates ferroptosis remain unclear. The primary aim of this study was to investigate how bromodomain-containing protein 4 affects neuronal ferroptosis following subarachnoid hemorrhage in vivo and in vitro. Our findings revealed that endogenous bromodomain-containing protein 4 co-localized with neurons, and its expression was decreased 48 hours after subarachnoid hemorrhage of the cerebral cortex in vivo. In addition, ferroptosis-related pathways were activated in vivo and in vitro after subarachnoid hemorrhage. Targeted inhibition of bromodomain-containing protein 4 in neurons increased lipid peroxidation and intracellular ferrous iron accumulation via ferritinophagy and ultimately led to neuronal ferroptosis. Using cleavage under targets and tagmentation analysis, we found that bromodomain-containing protein 4 enrichment in the Raf-1 promoter region decreased following oxyhemoglobin stimulation in vitro. Furthermore, treating bromodomain-containing protein 4-knockdown HT-22 cell lines with GW5074, a Raf-1 inhibitor, exacerbated neuronal ferroptosis by suppressing the Raf-1/ERK1/2 signaling pathway. Moreover, targeted inhibition of neuronal bromodomain-containing protein 4 exacerbated early and long-term neurological function deficits after subarachnoid hemorrhage. Our findings suggest that bromodomain-containing protein 4 may have neuroprotective effects after subarachnoid hemorrhage, and that inhibiting ferroptosis could help treat subarachnoid hemorrhage.

Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer.

To identify drivers of malignancy in human pancreatic ductal adenocarcinoma (PDAC), we performed regulatory network analysis on a large collection of expression profiles from laser capture microdissected samples of PDAC and benign precursors. We discovered that BMAL2 plays a role in the initiation, progression, post resection survival, and KRAS activity in PDAC. Functional analysis of BMAL2 target genes led us to hypothesize that it plays a role in regulating the response to hypoxia, a critical but poorly understood feature of PDAC physiology. Knockout of BMAL2 in multiple human PDAC cell lines revealed effects on viability and invasion, particularly under hypoxic conditions. Loss of BMAL2 also affected glycolysis and other metabolic processes. We found that BMAL2 directly regulates hypoxia-responsive target genes. We also found that BMAL2 is necessary for the stabilization of HIF1A upon exposure to hypoxia, but destabilizes HIF2A under hypoxia. These data demonstrate that BMAL2 is a master transcriptional regulator of hypoxia responses in PDAC and may serve as a long-sought molecular switch that distinguishes HIF1A- and HIF2A-dependent modes of hypoxic metabolism.

LGG-22. INVESTIGATING THE EFFICACY OF BELVARAFENIB, A TYPE II PAN-RAF INHIBITOR, AS A POTENTIAL THERAPEUTIC AGENT TO TREAT PEDIATRIC LOW-GRADE GLIOMAS HARBORINGBRAF FUSIONS

Abstract BACKGROUND Low-grade gliomas (LGGs) are the most common central nervous system (CNS) tumor found in children, comprising nearly one-third of all diagnoses. Fifteen to twenty percent of pediatric LGGs arise due to mutations in the RAS/mitogen-activated protein kinase (MAPK) signaling pathway, most commonly at the B-Raf proto-oncogene (BRAF) gene. Modern advances in personalized medicine have led to widespread use of targeted therapies to treat pediatric LGGs, specifically pan-RAF inhibitors that target BRAF V600E missense mutations and KIAA1549:BRAF fusions. However, to date, very few RAF inhibitors have been approved for use in children. METHODS The present study utilized patient-derived pediatric LGG cell lines to investigate the efficacy of Belvarafenib (0.5nM to 5nM), a type II pan-RAF inhibitor, as a potential therapeutic agent to treat BRAF-altered pediatric LGGs. A recent study demonstrated Belvarafenib’s ability to cross the blood-brain barrier, leading to an interest in its potential use in BRAF-mutated pediatric brain tumors. Belvarafenib has already shown promise in treating BRAF V600E-mutated melanomas and other solid tumors in adults, although its ability to effectively target BRAF fusions is unclear. Hence, three pediatric patient-derived cell lines with BRAF fusions will help elucidate any effect from Belvarafenib. RESULTS When treated with Belvarafenib, the cell lines harboring BRAF fusions demonstrated the compound’s inability to significantly decrease tumor cell viability. CONCLUSIONS This preliminary data highlights the possible limitations associated with using Belvarafenib as a single agent to treat pediatric LGGs with BRAF fusions.

LGG-40. TYPE II RAF INHIBITOR TOVORAFENIB IN RELAPSED/REFRACTORY PEDIATRIC LOW-GRADE GLIOMA (PLGG): REVERSIBLE DECREASES IN GROWTH VELOCITY IN THE PHASE 2 FIREFLY-1 TRIAL

Abstract BACKGROUND: Tovorafenib is an investigational, selective, CNS-penetrant, type II RAF inhibitor. The ongoing FIREFLY-1 (NCT04775485) phase 2 study (Kilburn LK, et al. Nat Med. 2023) of tovorafenib in BRAF-altered pLGG resulted in antitumor activity and manageable safety. Decreased growth velocity (GV) was observed; this is an update on GV changes in skeletally immature children receiving tovorafenib. Methods A planned safety analysis was completed on August 8, 2023 on 137 patients (Arm 1: 77 & Arm 2: 60). Additional follow-up on all cases of decreased GV (an AESI) reported to the global safety database (GSDB) as of January 19, 2024 is provided. Results Overall, 29% had decreased GV from baseline (BL); 19% had ≥50% decrease. Of the 40 with this AESI, 75% had pre-existing neuromuscular or endocrine comorbidities potentially affecting normal growth, including 6 on GnRH-analogues for precocious puberty and 9 with BL heights 2 SDs above/below average for age and sex. Nineteen had on-treatment bone age assessments; none showed bone age advancement from BL or premature growth plate closure. No osteopenia or abnormal fractures reported. All 10 who discontinued or interrupted tovorafenib for ≥3 months for any reason (mean follow-up: 5.8 months), with off-treatment growth measurements available, showed post-treatment annualized GV (AGV) recovery (average AGV: on-treatment, 1.1 cm/y; off-treatment, 8 cm/y), with some exceeding expected average AGV for age. A 4-year-old boy with 1.2 cm/y AGV on-treatment had 12.3 cm/y AGV off-treatment (follow-up: 2 months). Five additional events of decreased GV in patients not on study FIREFLY-1 were reported to the GSDB; 4 of 5 had ≥3 months of off-treatment follow-up, all 4 recovered GV. Conclusions Decreased GV has been observed in patients on tovorafenib. Preliminary follow-up data in those who interrupted treatment show consistent evidence of GV recovery and preservation of growth potential on bone age studies.

MicroRNA-focused CRISPR/Cas9 screen identifies miR-142 as a key regulator of Epstein-Barr virus reactivation.

Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle.

Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

The USP10/13 inhibitor, spautin-1, attenuates the progression of glioblastoma by independently regulating RAF-ERK mediated glycolysis and SKP2.

Glioblastoma is a malignant brain tumor with poor prognosis. Though several dysregulated pathways were found to mediate the tumor progression, hyperactivation of RAS-RAF-ERK pathway, enhanced glycolysis and SKP2 are associated with several glioblastomas. Recent findings on the role of USP10 in the transition from pro-neural to mesenchymal subtype of glioblastoma and, USP13 in the stabilization of RAF1 in mouse embryonic stem cells prompted us to examine their role in the mechanisms mediating the progression of glioblastoma. In the present study, we have examined the role of spautin-1, a pharmacological inhibitor of USP10 and USP13 in the mechanisms mediating glioblastoma. Our results indicate that spautin-1 as well as knockdown of its downstream targets, USP10 and USP13, reduced the proliferation and migration of glioblastoma cells. Also, spautin-1 mediated inhibition of RAF-ERK pathway or inhibition of RAF1 and MEK1 per se reduced the glycolytic function via PKM2/Glut-1 and inhibited the progression of glioblastoma. Further, the protooncogene, SKP2, which was shown to be a direct target of USP10 /USP13 was also reduced by spautin-1. While inhibition of SKP2 enhanced its downstream target p21, no apparent changes in the RAF-ERK levels or glycolytic function were evident. Also, inhibition of MEK1 did not affect SKP2 levels, indicating that these two pathways act independent of each other. Overall, our findings indicate that spautin-1 by virtue of its inhibitory effects on USP10/13 counteracts RAS-RAF-ERK mediated glycolysis and SKP2 that are critical in the progression of glioblastoma. Hence, further preclinical validation is warranted for taking the present observations forward.

A critical review of RAF inhibitors in BRAF-mutated glioma treatment.

BRAF gliomas have garnered significant attention in research due to the lack of effective treatments and their notable incidence, constituting 3% of all gliomas. This underlines the importance of investigating this area and the impact that targeted therapies could hold. This review discusses the development of targeted therapies for these tumors, examining the effectiveness of first-generation BRAF inhibitors such as Vemurafenib, Dabrafenib and Encorafenib, while addressing the challenges posed by paradoxical ERK activation. The advent of pan-RAF inhibitors, notably Tovorafenib, offers a promising advance, demonstrating enhanced efficacy and better penetration of the blood-brain barrier, without the issue of paradoxical activation. Nevertheless, continued research is essential to refine therapeutic strategies for BRAF-mutated gliomas, given the evolving nature of targeted therapy development.

An open-label study to assess the safety and efficacy of naporafenib (ERAS-254) administered with trametinib in previously treated patients with locally advanced unresectable or metastatic solid tumor malignancies with RAS Q61X mutations (SEACRAFT-1).

TPS3178 Background: The rat sarcoma viral oncogene (RAS)/mitogen-activated protein kinase (MAPK) pathway is a key signaling cascade that drives cell proliferation, differentiation, and survival. Inappropriate activation of this pathway drives tumorigenesis in a subset of solid tumors. There are 3 isoforms of RAS (KRAS, NRAS, and HRAS) with the most commonly mutated codons being 12, 13, and 61. While these codon mutations map to the active site of RAS, they appear to have differential impacts on pathway activation. Mutations at codon 61 (RAS Q61X) are generally thought to be the most MAPK pathway-activating relative to mutations at codons 12 and 13 and are therefore thought to be the most oncogenic. Naporafenib is a pan-RAF inhibitor that is being evaluated in combination with trametinib, which inhibits MEK, a downstream node of the RAS/MAPK pathway. Clinical proof of concept (PoC) in patients with NRASm melanoma (of which 80 to 90% have Q61X mutations) and preliminary PoC for patients with RAS Q61X NSCLC have been established for the combination. Preclinical data across tumor types suggest that RAS Q61X is a potential predictive biomarker for the combination of naporafenib with trametinib, a hypothesis being tested in this trial. Methods: SEACRAFT-1 (NCT05907304) is a Phase 1b, open-label study evaluating the clinical activity and safety of the combination of naporafenib and trametinib in pts with locally advanced, unresectable, or metastatic solid tumor malignancies that are not responsive to standard therapies or for which there are no standard therapies. Eligible pts must be ≥18 years (or ≥12 and <18 years in an adolescent substudy) with documented RAS Q61X mutation by an analytically validated assay. Pts must also have ≥1 measurable lesion per RECIST (Response Evaluation Criteria in Solid Tumors, v1.1) and Eastern Cooperative Oncology Group (ECOG) Performance Status ≤2 (or Karnofsky/Lansky Performance Status ≥70 for adolescent pts). Exclusion criteria include prior therapy with an ERK, MEK, RAF, or RAS inhibitor, QTcF >450 ms at baseline, and left ventricular ejection fraction (LVEF) <50% at baseline. A total of up to 115 pts will be enrolled to receive naporafenib 200 mg BID and trametinib 1 mg QD continuous in 28-day cycles until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint is ORR; secondary endpoints include assessment of PFS, DOR, safety, and tolerability. Clinical trial information: NCT05907304 .

A phase 1 study of the BET inhibitor ZEN003694 in combination with the MEK1/2 inhibitor binimetinib in solid tumors with RAS pathway alterations and triple-negative breast cancer (NCI number 10449).

TPS3182 Background: Bromodomain and extra-terminal (BET) proteins serve as epigenetic readers and regulate transcription by binding acetylated lysines in histones. BET inhibitors target this epigenetic machinery modifying the expression of oncogenes and have the potential to overcome drug resistance in different settings. Resistance to mitogen-activated extracellular kinase (MEK) inhibition appears to rely upon changes in gene expression, a process that can be potentially inhibited via BET blockade. Preclinical data have demonstrated synergistic efficacy of dual MEK and BET inhibition in MAPK-altered solid tumors. Triple negative breast cancer (TNBC) is a disease frequently found to have gene amplifications in BRAF, NF1 mutations, and upstream regulators of the MAPK pathway and preclinical work has shown efficacy with dual inhibition of MEK and BET. Methods: This phase 1, open-label, multicenter study was designed to evaluate the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of the BET inhibitor ZEN003694 in combination with the MEK inhibitor binimetinib in solid tumors with RAS pathway alterations and TNBC. The study has two parts: dose escalation (part 1) which plans to recruit a maximum of 30 patients and dose expansion (part 2), which will comprise 12 patients. The primary objective of part 1 is to determine the MTD and recommended Phase 2 dose (RP2D), whereas the primary objective of dose expansion is to evaluate safety and toxicity. Secondary objectives include tolerability, pharmacokinetics (PK), and antitumor activity. Additional analyses will explore epigenetic changes such as ChIPseq to determine levels of H3K27ac and ATACseq to determine treatment-induced changes in open chromatin regions. ZEN003694 is given orally daily, and binimetinib is administered orally twice daily in 28-day cycles. Dose escalation is being conducted using a standard 3+3 cohort design to determine the MTD. Part 1 requires evaluable or measurable disease, whereas Part 2 requires measurable disease as per RECIST v1.1. Patients must have an ECOG performance status of ≤2. Included tumors are TNBC (estrogen receptor ≤1%, progesterone receptor ≤1%, human epidermal growth factor receptor 2 (HER2) 0–1+ or non-amplified) or any other solid tumor with actionable MAPK alterations (e.g. KRAS, NRAS, HRAS, or BRAF activating mutations, inactivating NF1 mutations, or BRAF fusions). Patients with any PI3K pathway activating genomic alterations are excluded. Prior therapy with BET, RAF, MEK, or ERK inhibitor is not permitted. This study is currently ongoing in dose escalation. Clinical trial information: NCI number 10449.

Type II RAF inhibitor tovorafenib in relapsed/refractory pediatric low-grade glioma (pLGG): Reversible decreases in growth velocity in the phase 2 FIREFLY-1 trial.

10036 Background: Tovorafenib is an investigational, selective, CNS-penetrant, type II RAF inhibitor. Results from the ongoing FIREFLY-1 (NCT04775485) phase 2 study (1) in BRAF-altered pLGG showed clinically meaningful tumor responses and a manageable safety profile with tovorafenib monotherapy. Decreased growth velocity (GV) was observed; this is an update on changes in GV in skeletally immature children receiving tovorafenib. Methods: A planned safety analysis was completed on August 8, 2023 and included 137 patients (Arm 1: 77 & Arm 2: 60) treated with ≥1 dose of tovorafenib in FIREFLY-1. This report provides additional follow-up on all cases of decreased growth velocity (GV), an adverse event of special interest (AESI), reported to the Sponsor’s global safety database (GSDB) as of January 19, 2024. Results: Decreased GV was reported in 40 (29.2%) of 137 patients; 26 (19%) had GV reduction ≥50% from baseline (BL). Of those with decreased GV, 30 (75.0%) had pre-existing neuromuscular or endocrine comorbidities that may affect normal growth, including 6 (15%) with precocious puberty being treated with a gonadotropin-releasing hormone analogue, and 9 (22.5%) with BL heights 2 standard deviations above or below average for age and sex. Nineteen of the 40 patients with this AESI had on-treatment bone age assessments; none showed advancement of bone age from BL or premature closure of growth plates. No osteopenia or abnormal fractures were reported. Of the 35 patients with growth hormone (GH) testing, only 2 had signs of GH deficiency (both had optic pathway tumors; 1 had GH deficiency at BL, the other, a new tumor-associated GH deficiency). Post-treatment heights ≥3 months off treatment were reported for 10 of 40 patients who had interrupted or discontinued treatment for any reason (mean follow up: 5.8 months), including 2 (1.5%) permanent discontinuations and 3 (2.2%) interruptions due to decreased GV. All 10 showed post-treatment recovery in annualized GV (AGV) (average on-treatment AGV 1.1 cm/y vs. average post-treatment AGV 8 cm/y), in some cases exceeding expected average AGV for age. One additional 4-year-old boy with on-treatment AGV of 1.2 cm/y had an off-treatment AGV of 12.3 cm/y after 2 months of off-treatment follow-up. Decreased GV was reported to the GSDB for 5 (11.4%) of 44 patients who received above the maximum dose of tovorafenib used in FIREFLY-1 in an ongoing investigator-initiated study. Four of the 5 patients from this study had ≥3 months of off-treatment follow-up reported; all 4 showed evidence of GV recovery. Conclusions: Decreased GV has been observed in patients treated with tovorafenib. Early follow-up data from patients whose treatment was interrupted show consistent evidence for GV recovery and preservation of growth potential on bone age studies. 1. Kilburn LK, et al. Nat Med.2023. Clinical trial information: NCT04775485 .

RAF1 facilitates KIT signaling and serves as a potential treatment target for gastrointestinal stromal tumor.

The aberrant activation of RAS/RAF/MEK/ERK signaling is important for KIT mutation-mediated tumorigenesis of gastrointestinal stromal tumor (GIST). In this study, we found that inhibition of RAF1 suppresses the activation of both wild-type KIT and primary KIT mutations in GIST, with primary KIT mutations showing greater sensitivity. This suggests a positive feedback loop between KIT and RAF1, wherein RAF1 facilitates KIT signaling. We further demonstrated that RAF1 associates with KIT and the kinase activity of RAF1 is necessary for its contribution to KIT activation. Accordingly, inhibition of RAF1 suppressed cell survival, proliferation, and cell cycle progression in vitro mediated by both wild-type KIT and primary KIT mutations. Inhibition of RAF1 in vivo suppressed GIST growth in a transgenic mouse model carrying germline KIT/V558A mutation, showing a similar treatment efficiency as imatinib, the first-line targeted therapeutic drug of GIST, while the combination use of imatinib and RAF1 inhibitor further suppressed tumor growth. Acquisition of drug-resistant secondary mutation of KIT is a major cause of treatment failure of GIST following targeted therapy. Like wild-type KIT and primary KIT mutations, inhibition of RAF1 suppressed the activation of secondary KIT mutation, and the cell survival, proliferation, cell cycle progression in vitro, and tumor growth in vivo mediated by secondary KIT mutation. However, the activation of secondary KIT mutation is less dependent on RAF1 compared with that of primary KIT mutations. Taken together, our results revealed that RAF1 facilitates KIT signaling and KIT mutation-mediated tumorigenesis of GIST, providing a rationale for further investigation into the use of RAF1 inhibitors alone or in combination with KIT inhibitor in the treatment of GIST, particularly in cases resistant to KIT inhibitors.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.