Paradoxical Activation Research Articles

Pharmacological restriction of genomic binding sites redirects PU.1 pioneer transcription factor activity

Transcription factor (TF) DNA-binding dynamics govern cell fate and identity. However, our ability to pharmacologically control TF localization is limited. Here we leverage chemically driven binding site restriction leading to robust and DNA-sequence-specific redistribution of PU.1, a pioneer TF pertinent to many hematopoietic malignancies. Through an innovative technique, ‘CLICK-on-CUT&Tag’, we characterize the hierarchy of de novo PU.1 motifs, predicting occupancy in the PU.1 cistrome under binding site restriction. Temporal and single-molecule studies of binding site restriction uncover the pioneering dynamics of native PU.1 and identify the paradoxical activation of an alternate target gene set driven by PU.1 localization to second-tier binding sites. These transcriptional changes were corroborated by genetic blockade and site-specific reporter assays. Binding site restriction and subsequent PU.1 network rewiring causes primary human leukemia cells to differentiate. In summary, pharmacologically induced TF redistribution can be harnessed to govern TF localization, actuate alternate gene networks and direct cell fate.

BRAF Inhibition and UVB Light Synergistically Promote Mus musculus Papillomavirus 1-Induced Skin Tumorigenesis.

The development of keratinocytic skin tumors, presumably attributable to paradoxical activation of the MAPK pathway, represents a relevant side effect of targeted therapies with BRAF inhibitors (BRAFis). The role of cutaneous papillomavirus infection in BRAFi-associated skin carcinogenesis, however, is still inconclusive. Employing the Mus musculus papillomavirus 1 (MmuPV1) skin infection model, the impact of BRAFis and UVB exposure on papillomavirus induced skin tumorigenesis was investigated in immunocompetent FVB/NCrl mice. Systemic BRAF inhibition in combination with UVB light induced skin tumors in 62% of the MmuPV1-infected animals. In contrast, significantly fewer tumors were observed in the absence of either BRAF inhibition, UVB irradiation or virus infection, as demonstrated by lesional outgrowth in 20%, 5% and 0% of the mice, respectively. Combinatory exposure to BRAFis and UVB favored productive viral infection, which was shown by high numbers of MmuPV1 genome copies and E1^E4 spliced transcripts and an abundance of E6/E7 oncogene mRNA and viral capsid proteins. BRAF inhibition, but not viral infection or UVB light, activated ERK1/2, whereas γH2AX expression, inducible by UVB light, remained unaltered by BRAFis. These results provide experimental evidence that BRAF inhibition and UVB irradiation synergistically promote MmuPV1-induced skin tumor development in vivo. This indicates an alternative pathway by which papillomavirus skin infection may contribute to BRAFi-associated skin tumorigenesis.

Paradoxical SERCA dysregulation contributes to atrial fibrillation in a model of diet-induced obesity.

Obesity is a major risk factor for atrial fibrillation (AF) the most common serious cardiac arrhythmia, but the molecular mechanisms underlying diet-induced AF remain unclear. In this study, we subjected mice to a chronic high-fat diet and acute sympathetic activation ('two-hit' model) to study the mechanisms by which diet-induced obesity promotes AF. Surface electrocardiography revealed that diet-induced obesity and sympathetic activation synergize during intracardiac tachypacing to induce AF. At the cellular level, diet-induced obesity and acute adrenergic stimulation facilitate the formation of delayed afterdepolarizations in atrial myocytes, implicating altered Ca2+ dynamics as the underlying cause of AF. We found that diet-induced obesity does not alter the expression of major Ca2+-handling proteins in atria, including the sarcoplasmic reticulum Ca2+-ATPase (SERCA), a major component of beat-to-beat Ca2+ cycling in the heart. Paradoxically, obesity reduces phospholamban phosphorylation, suggesting decreased SERCA activity, yet atrial myocytes from obese mice showed a significantly increased Ca2+ transient amplitude and SERCA-mediated Ca2+ uptake. Adrenergic stimulation further increases the Ca2+ transient amplitude but does not affect Ca2+ reuptake in atrial myocytes from obese mice. Transcriptomics analysis showed that a high-fat diet prompts upregulation of neuronatin, a protein that has been implicated in obesity and is known to stimulate SERCA activity. We propose a mechanism in which obesity primes SERCA for paradoxical activation, and adrenergic stimulation facilitates AF conversion through a Ca2+-induced Ca2+ release gain in atrial myocytes. Overall, this study links obesity, altered Ca2+ signaling, and AF, and targeting this mechanism may prove effective for treating obesity-induced AF.

TRLS-02. SAFETY AND EFFICACY OF BRAF INHIBITOR PLIXORAFENIB (FORE8394; PLX8394) IN CHILDREN AND ADULTS WITH RECURRENT, BRAF-ALTERED PRIMARY CENTRAL NERVOUS SYSTEM TUMORS (PCNST)

Abstract BACKGROUND Plixorafenib is a selective, potent BRAFi that inhibits V600 and non-V600 alterations by disrupting BRAF monomers/dimers. Plixorafenib evades paradoxical MAPK pathway activation, eliminating the need for MEKi combination, and is potentially less prone to toxicities and resistance typical of approved BRAFis. METHODS Phase 1/2a study (NCT02428712) in children (n=5, aged 4-17 years) and adults (n=108) with BRAF-altered solid tumors assessing safety, PK, and efficacy of oral plixorafenib 900-3600mg/day ± cobicistat (PK enhancer). RESULTS Five children, 3 LGG (1 V600E, 1 fusion, 1 amplification), 1 neuroblastoma (G469A) and 1 ND-LCH (V600E), 4-8 prior therapies, received plixorafenib 300-1200 mg/day without cobicistat. Four received BSA-adjusted dosing, which delivered lower exposures than RP2D. All had SD, with duration 66.9+ months for ND-LCH. The fifth (neuroblastoma, 1200 mg/day) had PD. No DLTs, dose reductions, or LFT elevations. One had possibly related Grade 3 headache. Of 9 evaluable MAPKi-naïve adults with V600E PCNST, 4/5 with HGG and 2/4 with LGG/GNT responded; ORR 67% (95% CI: 29.9-92.5), mDOR 13.9 months. Responses in patients with only non-enhancing lesions and/or pilocytic astrocytoma indicate clinically relevant CNS penetration. Responses occurred across V600 solid tumors, ORR 42% (10/24); 1-year PFS 54.4%, 2-year PFS 48.4% in the MAPKi-naïve (non-CRC) subgroup. Of 14 evaluable adults with fusions, 2 responses (melanoma: CR, DOR 62.6+ months; PTC: PR, DOR 4.0+ months), 8 SD (5 with PCNST), 5 PD (3/5 with co-occurring driver mutations). Safety, efficacy, and PK in adults and children and insight into factors affecting plixorafenib exposure (not age or weight dependent) determined the optimal RP2D of plixorafenib as 900 mg QD with cobicistat for ages ≥10 years. CONCLUSIONS Plixorafenib had low rates of symptomatic TEAEs with a manageable safety profile, and high ORR in MAPKi-naïve BRAFV600 PCNST. A phase 2 study in children and adults is ongoing (NCT05503797).

Off-targets of BRAF inhibitors disrupt endothelial signaling and vascular barrier function.

Targeted therapies against mutant BRAF are effectively used in combination with MEK inhibitors (MEKi) to treat advanced melanoma. However, treatment success is affected by resistance and adverse events (AEs). Approved BRAF inhibitors (BRAFi) show high levels of target promiscuity, which can contribute to these effects. The blood vessel lining is in direct contact with high plasma concentrations of BRAFi, but effects of the inhibitors in this cell type are unknown. Hence, we aimed to characterize responses to approved BRAFi for melanoma in the vascular endothelium. We showed that clinically approved BRAFi induced a paradoxical activation of endothelial MAPK signaling. Moreover, phosphoproteomics revealed distinct sets of off-targets per inhibitor. Endothelial barrier function and junction integrity were impaired upon treatment with vemurafenib and the next-generation dimerization inhibitor PLX8394, but not with dabrafenib or encorafenib. Together, these findings provide insights into the surprisingly distinct side effects of BRAFi on endothelial signaling and functionality. Better understanding of off-target effects could help to identify molecular mechanisms behind AEs and guide the continued development of therapies for BRAF-mutant melanoma.

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 .

Functional validation of EIF2AK4 (GCN2) missense variants associated with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a disorder with a large genetic component. Biallelic mutations of EIF2AK4, which encodes the kinase GCN2, are causal in two ultra-rare subtypes of PAH, pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis. EIF2AK4 variants of unknown significance have also been identified in patients with classical PAH, though their relationship to disease remains unclear. To provide patients with diagnostic information and enable family testing, the functional consequences of such rare variants must be determined, but existing computational methods are imperfect. We applied a suite of bioinformatic and experimental approaches to sixteen EIF2AK4 variants that had been identified in patients. By experimentally testing the functional integrity of the integrated stress response (ISR) downstream of GCN2, we determined that existing computational tools have insufficient sensitivity to reliably predict impaired kinase function. We determined experimentally that several EIF2AK4 variants identified in patients with classical PAH had preserved function and are therefore likely to be non-pathogenic. The dysfunctional variants of GCN2 that we identified could be subclassified into three groups: misfolded, kinase-dead, and hypomorphic. Intriguingly, members of the hypomorphic group were amenable to paradoxical activation by a type-1½ GCN2 kinase inhibitor. This experiment approach may aid in the clinical stratification of EIF2AK4 variants and potentially identify hypomorophic alleles receptive to pharmacological activation.

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.

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

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

Abstract CT178: Phase 1 trial of PF-07799933 (ARRY-440), a next-generation BRAF inhibitor, for BRAF-mutant cancers

Abstract BACKGROUND Approved BRAF inhibitors (BRAFi) have transformed the treatment of BRAF V600-mutant cancers. However, clinical benefit is limited by BRAF dimer-promoting resistance mutations, toxicity from paradoxical activation of signaling in BRAF wild-type cells, and poor brain penetration. Further, current RAF inhibitors are ineffective against non-V600 BRAF mutants. Next-generation agents that cause pan-RAF inhibition may more broadly target BRAF mutants but are limited by a narrow therapeutic index. PF-07799933 (ARRY-440) is a brain-penetrant BRAF-selective monomer/dimer inhibitor that spares ARAF and CRAF. METHODS We characterized PF-07799933 in patient (pt)-derived BRAF-mutant cancer cells in vitro and in vivo. We investigated PF-07799933 in pts with refractory BRAF-mutant solid tumors using a novel first-in-patient phase 1 design that enabled flexible, rapid dose escalation based on safety and pharmacokinetics (PK) assessments. RESULTS PF-07799933 inhibited signaling in vitro, disrupted BRAF-containing homo- and heterodimers, and caused less paradoxical signaling than approved agents. PF-07799933 alone and in combination with binimetinib inhibited tumor growth systemically and in the brain in mouse xenografts harboring de novo and acquired BRAF dimer-forming mutations. As of August 24, 2023 data cutoff, 30 pts with BRAF-mutant cancers started PF-07799933 treatment at 50 mg QD-450 mg BID as monotherapy (60%) or combination with binimetinib (27%) or cetuximab (all colorectal cancer [CRC]) (13%). Tumor types were melanoma (mel) (43%), CRC (17%), primary brain (PBT) (13%), thyroid (10%), and other (n=1/3% each). BRAF mutations were class I (V600E) (73%), Class II (13%), and class III (13%). All BRAF V600E+ cancer pts were previously treated with ≥1 approved BRAFi and all BRAF V600E+ mel pts also were previously treated with ≥1 immune checkpoint inhibitor. PF-07799933 was well-tolerated as monotherapy or combination. There were no DLTs and the MTD was not reached. Treatment-emergent adverse events (TEAEs) in ≥3 pts for monotherapy were fatigue (any grade 44%/grade ≥3 0%), headache (28%/0%), vision blurred (22%/6%), and lipase increased (16%/0%). The most common TEAEs for combination were peripheral edema (33%/0%), acneiform rash, diarrhea, and fatigue (each 28%/0%). The AE profile was consistent with less paradoxical signaling activation. PK-guided dose escalation enabled PF-07799933 efficacious concentrations to be reached by dose level 3 (225 mg BID), together with multiple confirmed responses in BRAF V600E+ cancer pts (n=4/7 at 225 mg BID ± binimetinib, includes 1 complete response [CR] in a BRAF V600E+ PBT pt) systemically, in the brain, and all after failure of approved BRAF inhibitors. A 5th confirmed response (2nd BRAF V600E+ PBT pt) occurred after data cutoff. This includes, to our knowledge, the first evidence of efficacy in a BRAF V600E+ pt with a dimerizing BRAF splice variant. A BRAF G466E+ (class III) adenoid cystic carcinoma pt had a molecular CR in ctDNA. CONCLUSION PF-07799933 treatment was associated with anti-tumor activity against BRAF V600- and non-V600-mutant cancers preclinically and in treatment-refractory pts. The novel, rapid PK-informed dose escalation design provides a new paradigm for accelerating testing of next-generation targeted therapies early in clinical development. TRIAL REGISTRATION NUMBER NCT05355701 Citation Format: Rona Yaeger, Meredith McKean, Rizwan Haq, Joseph T. Beck, Matthew H. Taylor, Jonathan Cohen, Daniel Bowles, Shirish M. Gadgeel, Catalin Mihalcioiu, Kyriakos P. Papadopoulos, Eli L. Diamond, Keren B. Sturtz, Gang Feng, Stefanie K. Drescher, Micaela B. Reddy, Bhaswati Sengupta, Arnab K. Maity, Suzy A. Brown, Anurag Singh, Eric N. Brown, Brian R. Baer, Jim Wong, Tung-Chung Mou, Wen I. Wu, Dean Kahn, Sunyana Gadal, Neal Rosen, John J. Gaudino, Patrice A. Lee, Dylan P. Hartley, Stephen Michael Rothenberg. Phase 1 trial of PF-07799933 (ARRY-440), a next-generation BRAF inhibitor, for BRAF-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT178.

Phenibutan-an Illegal Food Supplement With Psychotropic Effects and Health Risks.

Phenibut (β-phenyl-γ-aminobutyric acid) is an analog of the neurotransmitter gamma-aminobutyric acid (GABA). Like abapentin and pregabalin, it inhibits α2-δ-subunits of voltagedependent presynaptic calcium channels. The potential harm resulting from the use of these gabapentinoids is currently a matter of debate. This review is based on pertinent publications retrieved by a selective literature search and on cases reported to the Giftinformationszentrum-Nord (GIZ-Nord), a poison information center at the University of Göttingen, Germany. Phenibut is a prescription drug in Russia but its production, possession, use, trafficking, or administration is illegal in Germany. The phenibut toxicity syndrome resembles that of gabapentinoids and GABA mimetics: benzodiazepine-like with - drawal symptoms including epileptic seizures, delirium and paradoxical activation have been described, as have cases of abuse and dependence. A few cases of use in the setting of multidrug abuse, and of phenibut-related death, have been described to date in the USA. The GIZ-Nord received 17 inquiries about phenibut, 55 about gabapentin, and 126 about pregabalin over the period 2008-2022. Over the same period, the GIZ-Nord was informed of 1207 cases involving Z substances and 4324 involving benzodiazepines. In the majority of the registered intoxications, including those with phenibut, the symptoms were mild. Overdoses of phenibut (2-100 g) were reported in 15 of the 17 cases; 8 of the persons who had taken an overdose were somnolent. In such cases, observation in intensive care was recommended. Respiratory depression or coma was not encountered in any case, not even in the patient who had taken 100 g of phenibut. Phenibut causes symptoms resembling those of gabapentinoid and benzodiazepine use. There have been reports of phenibut use in combination with other psychotropic drugs; in particular, its use together with opiates could increase the risk of coma and respiratory depression. No deaths due to phenibut intoxication have been published in Germany or elsewhere in Western Europe, although such cases may have been overlooked, as this drug is still largely unknown to Western medicine.

Paradoxical Activation of Oncogenic Signaling as a Cancer Treatment Strategy.

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress response programs that counteract the inherent toxicity of such aberrant signaling. Although inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of protein phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor-suppressive resistance. Significance: A therapy consisting of deliberate hyperactivation of oncogenic signaling combined with perturbation of the stress responses that result from this is very effective in animal models of colon cancer. Resistance to this therapy is associated with loss of oncogenic signaling and reduced oncogenic capacity, indicative of tumor-suppressive drug resistance.

Abstract 6489: Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations

Abstract We sought to screen all FDA approved, cancer-indicated, kinase targeting drugs using the gold standard radiometric biochemical activity assay (HotSpot) against the largest collection of kinases in the world (>100 drugs against 735 total kinases including 395 wild type and 340 mutants). One of these drugs, mitapivat, is an activator of Pyruvate Kinase. Here, we show mitapivat-driven biochemical inhibition of several, but not all, mutants of c-KIT. We also observed the equal and opposite phenomenon whereby many ATP-competitive kinase inhibitors demonstrate biochemical activation of certain kinases, several of which seem particularly susceptible to the effect. Previous work by other investigators (at Genentech and the Rosen lab, MSKCC) demonstrated that PLX4720 (precursor to Vemurafenib) paradoxically activated wild type BRAF, but not BRAF mutants, by driving dimerization of the kinase in drug concentrations insufficient to inhibit both members of the dimerized pair. Our data suggests this phenomenon may be more widespread than previously appreciated and fully observable in vitro. Such paradoxical activation could be driving both toxicities and inefficacy against cancers expressing some of these activatable kinases. This information could suggest novel opportunities for combination therapy and improve outcomes by contraindicating certain medication from use based on tumor expression profile. Citation Format: Christian M. Loch, Mia Eason, Shuguang Liang, Alison Goupil, Anthony Acinapura, Jamie Purcell, Maia Domiguez. Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations [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 6489.

Abstract 4609: Plixorafenib (plixo) synergizes with MEK inhibitors (MEKi) in MAPK pathway inhibition in BRAF V600 and non V600 alterations, with higher potency compared to early generation BRAFi and pan-RAFi

Abstract The efficacy of BRAFi in V600 mutated tumors is established and requires effective inhibition of the MAPK pathway. However, the use approved BRAFi leads to paradoxical MAPK pathway activation associated with toxicities and to acquired resistance. Therefore, these inhibitors were combined with MEKi to mitigate toxicities, which has been shown to be more effective than monotherapy in some settings. Although effective, these combinations result in bothersome side effects, a high discontinuation rate, and risk of secondary malignancies, indicating a continued unmet need. Also, ~35% of all BRAF mutations occur outside the V600 codon, composed of a wide array of missense mutations, fusions, and in-frame deletions, which are not targeted by the earlier generation BRAFi. By design, plixo (FORE8394; PLX8394), a clinical stage BRAFi, binds the monomeric form of BRAF and inhibits its dimerization, while sparing the WT form, avoiding paradoxical MAPK pathway activation. We hypothesized that the combination of plixo with a MEKi will improve its efficacy and potency through maximal suppression of the MAPK pathway. Using ForeSight assay, a unique platform for testing MAPK signaling, we tested the efficacy of plixo and 4 MEKi (trametinib, cobimetinib, binimetinib and mirdametinib) across a cohort 18 BRAF alterations (V600E, 2 class II and 15 fusions) as single agents and in combinations. The combinations of plixo with the different MEKi were synergistic across all BRAF alterations tested. Furthermore, we compared the potency of a combination of plixo with binimetinib (bini) to that of another BRAFi (vemurafenib) or pan-RAFi’s (tovorafenib and lifirafenib) with bini in 2 class I, 3 class II and 7 BRAF fusions using the foresight assay. The IC:50 of BRAF V600E expressing cells treated with plixo + bini was 6nM compared with 57nM, 103nM and 190nM for the tovorafenib, vemurafenib, and lifirafenib combinations with bini respectively. A similar trend was shown in 9 of 11 additional alterations tested, indicating the potency of the combination of plixo + bini. Results were validated using western blot analysis and cell viability assays of both melanoma and CRC V600E mutated cell lines. Western blot correlated with ForeSight results both in single agents and combination treatment. Cells treated with plixo + bini showed complete growth inhibition while the same drug concentrations of vemurafenib + bini and tovorafenib + binimetinib showed no growth inhibition beyond the effect of bini single agent. These results indicate the improved efficacy of plixo + bini combination in inhibiting MAPK signaling and cancer cell proliferation. Overall results support that maximal suppression of the MAPK pathway with plixo is more potent in combination with a MEK inhibitor in BRAF V600 and non-V600 nonclinical models compared to BRAF or pan-RAF combinations. Citation Format: Gabi Tarcic, Limor Cohen, Stacie Peacock Shepherd, Natalie Filippov Levy, Hana Billauer, Lea Birnbaum, Sara Gasasa, Roey Maor, Reham Abu-Liel, Eden Goldfarb. Plixorafenib (plixo) synergizes with MEK inhibitors (MEKi) in MAPK pathway inhibition in BRAF V600 and non V600 alterations, with higher potency compared to early generation BRAFi and pan-RAFi [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 4609.

Abstract 1658: CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis

Abstract Activating mutations in BRAF at residue V600 (typically V600E) lead to dysregulation of the MAPK pathway and occur in approximately 8% of all human cancers including 60% of melanoma, up to 12% of colorectal cancer (CRC), and 4% of NSCLC. Currently approved BRAF inhibitors (BRAFi) are selective for BRAF V600X mutant proteins and are typically used in combination with MEK inhibitors (MEKi) in melanoma and NSCLC, or anti-EGFR antibodies such as cetuximab in CRC. However, their activity is limited by primary or acquired resistance often mediated by RAF dimer-inducing mechanisms. Furthermore, progression of BRAF V600X melanoma after BRAFi/MEKi treatment frequently involves brain metastasis, and currently approved BRAFi have relatively poor brain penetration. CFT1946 is a potent, orally bioavailable, cereblon-based BiDACTM degrader that selectively degrades BRAF V600X mutant protein and is currently under investigation in a Phase I clinical trial. CFT1946 has the potential to overcome limitations of clinically approved BRAFi, as degradation of BRAF V600X should abrogate RAF dimer-driven resistance and paradoxical activation. Indeed, we have previously demonstrated that CFT1946 is efficacious in an A375 BRAF V600E/NRAS Q61K xenograft model of BRAFi resistant melanoma. Here we substantially expand our preclinical characterization of CFT1946 across multiple models of BRAF V600X-driven cancers including BRAF V600X-driven CRC and NSCLC, additional BRAFi-resistant melanoma models, and a brain metastatic melanoma model. Single agent CFT1946 outperformed the SOC encorafenib + cetuximab combination in a panel of BRAF-V600X CRC xenograft models. Pathway analysis in BRAF V600X CRC models revealed that CFT1946 suppresses EGFR-mediated MAPK pathway reactivation, a mechanism known to diminish the impact of approved BRAF inhibitors in this indication. CFT1946 also demonstrated regression in a BRAF-V600X NSCLC PDX model where the SOC dabrafenib + trametinib showed only modest tumor growth inhibition. Consistent with our previous results in the A375 melanoma model, single agent CFT1946 showed superior activity versus the SOC dabrafenib + trametinib combination in all additional melanoma models tested, with complete regression achieved using a CFT1946 + trametinib combination in a PDX model bearing a BRAF-V600E kinase duplication that showed minimal response to dabrafenib + trametinib. Finally, using an A375 intracranial model, treatment with CFT1946 gave robust, dose dependent efficacy and survival advantage over encorafenib. The promising activity of CFT1946 in a broad range of BRAF V600X preclinical models supports its ongoing clinical investigation in BRAF V600 mutant solid tumors (NCT05668585). Citation Format: Bridget Kreger, Jacob R. Stephenson, Mathew E. Sowa, Samantha A. Perino, Laura L. Poling, Eunju Hurh, Michael J. Thomenius, Yanke Liang, Stewart L. Fisher, Roy M. Pollock. CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis [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 1658.

Abstract 1947: Studies on pMEK inhibitors for the treatment of RAS-mutant cancers as a single agent or combinations

Abstract RAS mutations are the most common oncogene in human cancers and KRAS has the highest frequency in non-small cell lung cancer (NSCLC) and pancreatic cancer (PDAC). KRAS G12C inhibitors have been approved to treat NSCLC patients. However, drug-resistance is eventually developed for these treatments. Preclinical studies suggested MAPK pathway reactivation is one possible mechanism. Among many MEK inhibitors in clinical development, CH5126766 binds to MEK1 inactive conformation and blocks its phosphorylation and activation. We believe this compound should prevent MAPK “paradoxical activation” commonly observed in most RAF and MEK inhibitors such as vemurafenib and cobimetinib. We studied this compound in various in vitro assays and found its potential in combating the paradoxical activation, which might be the cause of drug-resistance for MAPK signal pathway inhibitors, including RAS-, RAF- MEK- and ERK-inhibitors, as a single agent or in combination with above-mentioned inhibitors. In addition, we are developing a series of compounds based on CH5126766, by improving the inhibition of MEK1/2 kinase activity in addition to its activation. In summary, studies on the pMEK inhibitor demonstrated that this class of compounds might mitigate the MAPK pathway reactivation and prolong drug efficacies in clinical. Detailed research results will be discussed. Citation Format: Xiaobing Lv, Min Xu, Yong Hu, Lanjiao Zhao, Xiuquan Chen, Youqin Chen, Chen Yang, Jizhi Li, Chen Chen. Studies on pMEK inhibitors for the treatment of RAS-mutant cancers as a single agent or combinations [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 1947.

Abstract 2499: Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS signaling

Abstract KRAS is mutated in ~85% of pancreatic ductal adenocarcinoma (PDAC) patient's tumors, suggesting that it is a key node of PDAC targeted therapy. RAF/MEK/ERK signaling is downstream of KRAS signaling. Preclinical studies targeting MEK showed promises but clinical trials failed to show the benefit compared to Standard-of-Care treatment chemotherapy. To elucidate the mechanism of action and resistance to several targeted therapies including MEK inhibition in human PDAC, we conducted a Window-Of-Opportunity trial for Metastatic pancreatic cancer (WOOM trial). In this trial, metastatic PDAC patients received 10 days of Cobimetinib, a FDA approved MEK inhibitor, therapy and specimens of pre- and post- treatment were obtained by biopsy to evaluate the response to targeted treatment using deep multi-omic analytics including DNA-seq, RNA-seq, multiplex IHC, cyclic IF and Digital Spatial Profiling (DSP). The clinical grade KI67 staining positivity was used as a readout of tumor response. We found three of 15 patient’s tumors had significantly decreased KI67 staining positivity after Cobimetinib treatment and defined them as Responsive tumors. Genomic sequence showed that KRAS allelic imbalance and KrasG12R mutations were seen significantly higher frequency in Responsive tumors compared to Non-responsive tumors. RNA-seq analysis showed that these Responsive tumors were more likely to be classical-like subtype compared to Non-responsive tumors. DSP analysis confirmed the downregulation of ERK phosphorylation after treatment in all examined cases consistent with effective target inhibition. Interestingly, MEK phosphorylation was upregulated after treatment paradoxically. This suggests that paradoxical upstream pathway activation including RAF activation could be potential resistance mechanism. MYC protein expression change was significantly correlated with KI67 protein expression change, which suggests MYC downregulation could contribute to action of Cobimetinib. We also found that Cobimetinib treatment increased the immunosuppressive TREM2+ Macrophage population from single cell RNA-seq data. Furthermore, PDX from biopsy recapitulated patient's response to therapy and could be a great tool for testing new therapeutic strategies. Here, we summarize the multi-omics data and discuss potential mechanisms of response and resistance to targeting RAS signaling and related microenvironmental changes. Citation Format: Motoyuki Tsuda, Colin Daniel, Xiaoyan Wang, Carl Pelz, Hayley Zimny, Alexander Smith, John Muschler, Xi Li, Tugba Yildiran Ozmen, Furkan Ozmen, Dove Keith, Christopher Corless, Koei Chin, Jonathan Brody, Charles Lopez, Gordon Mills, Rosalie Sears. Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS signaling [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 2499.

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 4588: Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient pancreatic cancer

Abstract KRAS is mutationally activated in 95% of pancreatic ductal adenocarcinoma (PDAC) patients. Direct KRAS inhibitors are under intense preclinical and clinical development, with two KRASG12C mutant-selective inhibitors (G12Ci) now approved. However, treatment-associated resistance to KRAS inhibitors has been reported in the clinic highlighting an urgent need to identify both additional therapeutic targets and novel combination treatment strategies with KRAS inhibitors. To that end, we performed a CRISPR/Cas9 loss-of-function screen using a library targeting ~2,500 druggable genes in KRASG12C/D-mutant PDAC cell lines. We identified multiple genes that were essential for PDAC cell growth and selected PRMT5, a protein arginine methyltransferase, to assess as a novel therapeutic target for pancreatic cancer. We found that suppression of PRMT5 activity using two distinct first-generation clinical candidate small molecule inhibitors (JNJ-64619178 and GSK3326595) demonstrated single agent activity and reduced PDAC cell growth. We further assessed a mechanistically distinct MTA-cooperative clinical candidate PRMT5 inhibitor, MRTX1719, that is selective for MTAP-deleted tumors, which occur in ~25% of PDAC patients. We determined that MRTX1719 exhibited low nanomolar GI50 activities in MTAP-deficient but not MTAP wild-type KRAS-mutant cell lines. Unexpectedly, PDAC cells treated with MRTX1719 exhibited a paradoxical activation of ERK, suggesting that combined inhibition of PRMT5 and KRAS might be therapeutically advantageous. In support of our hypothesis, we demonstrated that combination treatment with MRTX1719 and mutant selective KRAS inhibitors (G12Ci/MRTX849 and G12Di/MRTX1133) further sensitized KRASG12C/D-mutant PDAC cells to KRAS inhibition in short- and long-term growth assays as well as in vivo xenograft studies. Our ongoing studies are evaluating the consequences of co-targeting PRMT5 and KRAS on gene expression changes using RNA-Seq and cancer cell signaling pathways using RPPA analyses. In summary, our data support concurrent inhibition of PRMT5 and KRAS as a promising therapeutic strategy for MTAP-deficient KRAS-mutant pancreatic cancer. Citation Format: Kristina Drizyte-Miller, Wen-Hsuan Chang, Andrew M. Waters, Clint A. Stalnecker, Junning Wang, Andrew J. Aguirre, Adrienne D. Cox, Channing J. Der. Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient pancreatic cancer [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 4588.

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.