RAF and MEK Inhibitors in Non-Small Cell Lung Cancer.

Lung cancer, despite recent advancements in survival rates, represents a significant global health burden. Non-small cell lung cancer (NSCLC), the most prevalent type, is driven largely by activating mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and receptor tyrosine kinases (RTKs), and less in v-RAF murine sarcoma viral oncogene homolog B (BRAF) and mitogen-activated protein-kinase kinase (MEK), all key components of the RTK-RAS-mitogen-activated protein kinase (MAPK) pathway. Learning from melanoma, the identification of BRAFV600E substitution in NSCLC provided the rationale for the investigation of RAF and MEK inhibition as a therapeutic strategy. The regulatory approval of two RAF-MEK inhibitor combinations, dabrafenib-trametinib, in 2017, and encorafenib-binimetinib, in 2023, signifies a breakthrough for the management of BRAFV600E-mutant NSCLC patients. However, the almost universal emergence of acquired resistance limits their clinical benefit. New RAF and MEK inhibitors, with distinct biochemical characteristics, are in preclinical and clinical development. In this review, we aim to provide valuable insights into the current state of RAF and MEK inhibition in the management of NSCLC, fostering a deeper understanding of the potential impact on patient outcomes.

Mechanism of Dimer Selectivity and Binding Cooperativity of BRAF Inhibitors.

Aberrant signaling of BRAFV600E is a major cancer driver. Current FDA-approved RAF inhibitors selectively inhibit the monomeric BRAFV600E and suffer from tumor resistance. Recently, dimer-selective and equipotent RAF inhibitors have been developed; however, the mechanism of dimer selectivity is poorly understood. Here, we report extensive molecular dynamics (MD) simulations of the monomeric and dimeric BRAFV600E in the apo form or in complex with one or two dimer-selective (PHI1) or equipotent (LY3009120) inhibitor(s). The simulations uncovered the unprecedented details of the remarkable allostery in BRAFV600E dimerization and inhibitor binding. Specifically, dimerization retrains and shifts the αC helix inward and increases the flexibility of the DFG motif; dimer compatibility is due to the promotion of the αC-in conformation, which is stabilized by a hydrogen bond formation between the inhibitor and the αC Glu501. A more stable hydrogen bond further restrains and shifts the αC helix inward, which incurs a larger entropic penalty that disfavors monomer binding. This mechanism led us to propose an empirical way based on the co-crystal structure to assess the dimer selectivity of a BRAFV600E inhibitor. Simulations also revealed that the positive cooperativity of PHI1 is due to its ability to preorganize the αC and DFG conformation in the opposite protomer, priming it for binding the second inhibitor. The atomically detailed view of the interplay between BRAF dimerization and inhibitor allostery as well as cooperativity has implications for understanding kinase signaling and contributes to the design of protomer selective RAF inhibitors.

Perspectives in Melanoma: meeting report from the Melanoma Bridge (December 1st-3rd, 2022-Naples, Italy).

Outcomes for patients with melanoma have improved over the past decade with the clinical development and approval of immunotherapies targeting immune checkpoint receptors such as programmed death-1 (PD-1), programmed death ligand 1 (PD-L1) or cytotoxic T lymphocyte antigen-4 (CTLA-4). Combinations of these checkpoint therapies with other agents are now being explored to improve outcomes and enhance benefit-risk profiles of treatment. Alternative inhibitory receptors have been identified that may be targeted for anti-tumor immune therapy, such as lymphocyte-activation gene-3 (LAG-3), as have several potential target oncogenes for molecularly targeted therapy, such as tyrosine kinase inhibitors. Unfortunately, many patients still progress and acquire resistance to immunotherapy and molecularly targeted therapies. To bypass resistance, combination treatment with immunotherapies and single or multiple TKIs have been shown to improve prognosis compared to monotherapy. The number of new combinations treatment under development for melanoma provides options for the number of patients to achieve a therapeutic benefit. Many diagnostic and prognostic assays have begun to show clinical applicability providing additional tools to optimize and individualize treatments. However, the question on the optimal algorithm of first- and later-line therapies and the search for biomarkers to guide these decisions are still under investigation. This year, the Melanoma Bridge Congress (Dec 1st-3rd, 2022, Naples, Italy) addressed the latest advances in melanoma research, focusing on themes of paramount importance for melanoma prevention, diagnosis and treatment. This included sessions dedicated to systems biology on immunotherapy, immunogenicity and gene expression profiling, biomarkers, and combination treatment strategies.

Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets.

Proteolysis Targeting Chimeras (PROTACs) are attractive therapeutic modalities for degrading disease-causing proteins. While many PROTACs have been developed for numerous protein targets, current small-molecule PROTAC approaches cannot target undruggable proteins that do not have small-molecule binders. Here, we present a novel PROTAC approach, termed bridged PROTAC, which utilizes a small-molecule binder of the target protein's binding partner to recruit the protein complex into close proximity with an E3 ubiquitin ligase to target undruggable proteins. Applying this bridged PROTAC strategy, we discovered MS28, the first-in-class degrader of cyclin D1, which lacks a small-molecule binder. MS28 effectively degrades cyclin D1, with faster degradation kinetics and superior degradation efficiency than CDK4/6, through recruiting the CDK4/6-cyclin D1 complex to the von Hippel-Lindau E3 ligase. MS28 also suppressed the proliferation of cancer cells more effectively than CDK4/6 inhibitors and degraders. Altogether, the bridged PROTAC strategy could provide a generalizable platform for targeting undruggable proteins.

CDK4: a master regulator of the cell cycle and its role in cancer.

The cell cycle is regulated in part by cyclins and their associated serine/threonine cyclin-dependent kinases, or CDKs. CDK4, in conjunction with the D-type cyclins, mediates progression through the G1 phase when the cell prepares to initiate DNA synthesis. Although Cdk4-null mutant mice are viable and cell proliferation is not significantly affected in vitro due to compensatory roles played by other CDKs, this gene plays a key role in mammalian development and cancer. This review discusses the role that CDK4 plays in cell cycle control, normal development and tumorigenesis as well as the current status and utility of approved small molecule CDK4/6 inhibitors that are currently being used as cancer therapeutics.

Perspectives in Melanoma: meeting report from the Melanoma Bridge (December 2nd - 4th, 2021, Italy).

Advances in immune checkpoint and combination therapy have led to improvement in overall survival for patients with advanced melanoma. Improved understanding of the tumor, tumor microenvironment and tumor immune-evasion mechanisms has resulted in new approaches to targeting and harnessing the host immune response. Combination modalities with other immunotherapy agents, chemotherapy, radiotherapy, electrochemotherapy are also being explored to overcome resistance and to potentiate the immune response. In addition, novel approaches such as adoptive cell therapy, oncogenic viruses, vaccines and different strategies of drug administration including sequential, or combination treatment are being tested. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic theràapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers, but they have yet to be fully characterized and implemented clinically. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. Overall, the future research efforts in melanoma therapeutics and translational research should focus on several aspects including: (a) developing robust biomarkers to predict efficacy of therapeutic modalities to guide clinical decision-making and optimize treatment regimens, (b) identifying mechanisms of therapeutic resistance to immune checkpoint inhibitors that are potentially actionable, (c) identifying biomarkers to predict therapy-induced adverse events, and (d) studying mechanism of actions of therapeutic agents and developing algorithms to optimize combination treatments. During the Melanoma Bridge meeting (December 2nd-4th, 2021, Naples, Italy) discussions focused on the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine as well as the impact of COVID-19 pandemic on management of melanoma patients.

Triple MAPK inhibition salvaged a relapsed post-BCMA CAR-T cell therapy multiple myeloma patient with a BRAF V600E subclonal mutation.

BACKGROUND: Multiple Myeloma (MM) is a progressive plasma cell neoplasm characterized by heterogeneous clonal expansion. Despite promising response rates achieved with anti-BCMA CAR-T cell therapy, patients may still relapse and there are currently no clear therapeutic options in post-CAR-T settings. In this report, we present a case of a post-BCMA CAR-T relapsed/refractory (RR) MM patient with skin extramedullary disease (EMD) in which a novel MAPK inhibition combinatorial strategy was implemented based on next-generation sequencing and in vitro experiments. CASE PRESENTATION: A 61-year-old male with penta-refractory MM penta- (IgA lambda), ISS stage 3 with hyperdiploidy, gain of 1q21 and del13 was treated with anti-BCMA CAR-T cell therapy, achieving a best response of VGPR. He progressed after 6 months and was salvaged for a short period with autologous stem cell transplantation. Eventually, he progressed with extramedullary disease manifested as subcutaneous nodules. Based on whole-exome sequencing, we identified a BRAF (V600E) dominant subclone in both bone marrow and cutaneous plasmacytoma. Following in vitro experiments, and according to our previous studies, we implemented a triple MAPK inhibition strategy under which the patient achieved a very good partial response for 110 days, which allowed to bridge him to subsequent clinical trials and eventually achieve a stringent complete response (sCR). CONCLUSION: Here, we show the applicability, effectiveness, and tolerability the triple MAPK inhibition strategy in the context of post-BCMA CAR-T failure in specific subset of patients. The triple therapy could bridge our hospice bound RRMM patient with BRAF (V600E) to further therapeutic options where sCR was achieved. We will further evaluate triple MAPK inhibition in patients with BRAF V600E in a precision medicine clinical trial launching soon.

Molecular Pathways and Mechanisms of BRAF in Cancer Therapy.

With the identification of activating mutations in BRAF across a wide variety of malignancies, substantial effort was placed in designing safe and effective therapeutic strategies to target BRAF. These efforts have led to the development and regulatory approval of three BRAF inhibitors as well as five combinations of a BRAF inhibitor plus an additional agent(s) to manage cancer such as melanoma, non-small cell lung cancer, anaplastic thyroid cancer, and colorectal cancer. To date, each regimen is effective only in patients with tumors harboring BRAFV600 mutations and the duration of benefit is often short-lived. Further limitations preventing optimal management of BRAF-mutant malignancies are that treatments of non-V600 BRAF mutations have been less profound and combination therapy is likely necessary to overcome resistance mechanisms, but multi-drug regimens are often too toxic. With the emergence of a deeper understanding of how BRAF mutations signal through the RAS/MAPK pathway, newer RAF inhibitors are being developed that may be more effective and potentially safer and more rational combination therapies are being tested in the clinic. In this review, we identify the mechanics of RAF signaling through the RAS/MAPK pathway, present existing data on single-agent and combination RAF targeting efforts, describe emerging combinations, summarize the toxicity of the various agents in clinical testing, and speculate as to where the field may be headed.

ROCK1 mechano-signaling dependency of human malignancies driven by TEAD/YAP activation.

Rho family mechano-signaling through the actin cytoskeleton positively regulates physiological TEAD/YAP transcription, while the evolutionarily conserved Hippo tumor suppressor pathway antagonizes this transcription through YAP cytoplasmic localization/degradation. The mechanisms responsible for oncogenic dysregulation of these pathways, their prevalence in tumors, as well as how such dysregulation can be therapeutically targeted are not resolved. We demonstrate that p53 DNA contact mutants in human tumors, indirectly hyperactivate RhoA/ROCK1/actomyosin signaling, which is both necessary and sufficient to drive oncogenic TEAD/YAP transcription. Moreover, we demonstrate that recurrent lesions in the Hippo pathway depend on physiological levels of ROCK1/actomyosin signaling for oncogenic TEAD/YAP transcription. Finally, we show that ROCK inhibitors selectively antagonize proliferation and motility of human tumors with either mechanism. Thus, we identify a cancer driver paradigm and a precision medicine approach for selective targeting of human malignancies driven by TEAD/YAP transcription through mechanisms that either upregulate or depend on homeostatic RhoA mechano-signaling.

Distinct CDK6 complexes determine tumor cell response to CDK4/6 inhibitors and degraders.

CDK4/6 inhibitors (CDK4/6i) are effective in metastatic breast cancer, but they have been only modestly effective in most other tumor types. Here we show that tumors expressing low CDK6 rely on CDK4 function, and are exquisitely sensitive to CDK4/6i. In contrast, tumor cells expressing both CDK4 and CDK6 have increased reliance on CDK6 to ensure cell cycle progression. We discovered that CDK4/6i and CDK4/6 degraders potently bind and inhibit CDK6 selectively in tumors in which CDK6 is highly thermo-unstable and strongly associated with the HSP90/CDC37 complex. In contrast, CDK4/6i and CDK4/6 degraders are ineffective in antagonizing tumor cells expressing thermostable CDK6, due to their weaker binding to CDK6 in these cells. Thus, we uncover a general mechanism of intrinsic resistance to CDK4/6i and CDK4/6i-derived degraders and the need for novel inhibitors targeting the CDK4/6i-resistant, thermostable form of CDK6 for application as cancer therapeutics.

AKT Degradation Selectively Inhibits the Growth of PI3K/PTEN Pathway-Mutant Cancers with Wild-Type KRAS and BRAF by Destabilizing Aurora Kinase B.

Using a panel of cancer cell lines, we characterized a novel degrader of AKT, MS21. In mutant PI3K-PTEN pathway cell lines, AKT degradation was superior to AKT kinase inhibition for reducing cell growth and sustaining lower signaling over many days. AKT degradation, but not kinase inhibition, profoundly lowered Aurora kinase B (AURKB) protein, which is known to be essential for cell division, and induced G2-M arrest and hyperploidy. PI3K activated AKT phosphorylation of AURKB on threonine 73, which protected it from proteasome degradation. A mutant of AURKB (T73E) that mimics phosphorylation and blocks degradation rescued cells from growth inhibition. Degrader-resistant lines were associated with low AKT phosphorylation, wild-type PI3K/PTEN status, and mutation of KRAS/BRAF. Pan-cancer analysis identified that 19% of cases have PI3K-PTEN pathway mutation without RAS pathway mutation, suggesting that these patients with cancer could benefit from AKT degrader therapy that leads to loss of AURKB. SIGNIFICANCE: MS21 depletes cells of phosphorylated AKT (pAKT) and a newly identified AKT substrate, AURKB, to inhibit tumor growth in mice. MS21 is superior to prior agents that target PI3K and AKT due to its ability to selectively target active, pAKT and sustain repression of signaling to deplete AURKB. This article is highlighted in the In This Issue feature, p. 2945.

BRAFV600E-induced senescence drives Langerhans cell histiocytosis pathophysiology.

Langerhans cell histiocytosis (LCH) is a potentially fatal condition characterized by granulomatous lesions with characteristic clonal mononuclear phagocytes (MNPs) harboring activating somatic mutations in mitogen-activated protein kinase (MAPK) pathway genes, most notably BRAFV600E. We recently discovered that the BRAFV600E mutation can also affect multipotent hematopoietic progenitor cells (HPCs) in multisystem LCH disease. How the BRAFV600E mutation in HPCs leads to LCH is not known. Here we show that enforced expression of the BRAFV600E mutation in early mouse and human multipotent HPCs induced a senescence program that led to HPC growth arrest, apoptosis resistance and a senescence-associated secretory phenotype (SASP). SASP, in turn, promoted HPC skewing toward the MNP lineage, leading to the accumulation of senescent MNPs in tissue and the formation of LCH lesions. Accordingly, elimination of senescent cells using INK-ATTAC transgenic mice, as well as pharmacologic blockade of SASP, improved LCH disease in mice. These results identify senescent cells as a new target for the treatment of LCH.

Exploiting Allosteric Properties of RAF and MEK Inhibitors to Target Therapy-Resistant Tumors Driven by Oncogenic BRAF Signaling.

Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF) but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF-MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAFV600E therapy-resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical well-being in a patient with stage IV colorectal cancer. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAFV600E tumors. SIGNIFICANCE: This work identifies a new class of RAFi that are selective for dBRAF over mBRAF and determines the basis of their selectivity. A rationally designed combination of RAF and MEK inhibitors based on their conformation selectivity achieved increased efficacy and a high therapeutic index when used to target BRAFV600E tumors.See related commentary by Zhang and Bollag, p. 1620.This article is highlighted in the In This Issue feature, p. 1601.

Inhibitors of BRAF dimers using an allosteric site.

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.

Global view of the RAF-MEK-ERK module and its immediate downstream effectors.

Small molecule inhibitors of BRAF and MEK have proven effective at inhibiting tumor growth in melanoma patients, however this efficacy is limited due to the almost universal development of drug resistance. To provide advanced insight into the signaling responses that occur following kinase inhibition we have performed quantitative (phospho)-proteomics of human melanoma cells treated with either dabrafenib, a BRAF inhibitor; trametinib, a MEK inhibitor or SCH772984, an ERK inhibitor. Over nine experiments we identified 7827 class I phosphorylation sites on 4960 proteins. This included 54 phosphorylation sites that were significantly down-modulated after exposure to all three inhibitors, 34 of which have not been previously reported. Functional analysis of these novel ERK targets identified roles for them in GTPase activity and regulation, apoptosis and cell-cell adhesion. Comparison of the results presented here with previously reported phosphorylation sites downstream of ERK showed a limited degree of overlap suggesting that ERK signaling responses may be highly cell line and cue specific. In addition we identified 26 phosphorylation sites that were only responsive to dabrafenib. We provide further orthogonal experimental evidence for 3 of these sites in human embryonic kidney cells over-expressing BRAF as well as further computational insights using KinomeXplorer. The validated phosphorylation sites were found to be involved in actin regulation, which has been proposed as a novel mechanism for inhibiting resistance development. These results would suggest that the linearity of the BRAF-MEK-ERK module is at least context dependent.

SHP2 Drives Adaptive Resistance to ERK Signaling Inhibition in Molecularly Defined Subsets of ERK-Dependent Tumors.

Pharmacologic targeting of components of ERK signaling in ERK-dependent tumors is often limited by adaptive resistance, frequently mediated by feedback-activation of RTK signaling and rebound of ERK activity. Here, we show that combinatorial pharmacologic targeting of ERK signaling and the SHP2 phosphatase prevents adaptive resistance in defined subsets of ERK-dependent tumors. In each tumor that was sensitive to combined treatment, p(Y542)SHP2 induction was observed in response to ERK signaling inhibition. The strategy was broadly effective in TNBC models and tumors with RAS mutations at G12, whereas tumors with RAS(G13D) or RAS(Q61X) mutations were resistant. In addition, we identified a subset of BRAF(V600E) tumors that were resistant to the combined treatment, in which FGFR was found to drive feedback-induced RAS activation, independently of SHP2. Thus, we identify molecular determinants of response to combined ERK signaling and SHP2 inhibition in ERK-dependent tumors.

Mouse ER+/PIK3CAH1047R breast cancers caused by exogenous estrogen are heterogeneously dependent on estrogen and undergo BIM-dependent apoptosis with BH3 and PI3K agents.

Estrogen dependence is major driver of ER + breast cancer, which is associated with PI3K mutation. PI3K inhibition (PI3Ki) can restore dependence on ER signaling for some hormone therapy-resistant ER + breast cancers, but is ineffective in others. Here we show that short-term supplementation with estrogen strongly enhanced Pik3caH1047R-induced mammary tumorigenesis in mice that resulted exclusively in ER + tumors, demonstrating the cooperation of the hormone and the oncogene in tumor development. Similar to human ER + breast cancers that are endocrine-dependent or endocrine-independent at diagnosis, tumor lines from this model retained ER expression but were sensitive or resistant to hormonal therapies. PI3Ki did not induce cell death but did cause upregulation of the pro-apoptotic gene BIM. BH3 mimetics or PI3Ki were unable to restore hormone sensitivity in several resistant mouse and human tumor lines. Importantly however, combination of PI3Ki and BH3 mimetics had a profound, BIM-dependent cytotoxic effect in PIK3CA-mutant cancer cells while sparing normal cells. We propose that addition of BH3 mimetics offers a therapeutic strategy to markedly improve the cytotoxic activity of PI3Ki in hormonal therapy-resistant and ER-independent PIK3CA-mutant breast cancer.

New perspectives for targeting RAF kinase in human cancer.

The discovery that a subset of human tumours is dependent on mutationally deregulated BRAF kinase intensified the development of RAF inhibitors to be used as potential therapeutics. The US Food and Drug Administration (FDA)-approved second-generation RAF inhibitors vemurafenib and dabrafenib have elicited remarkable responses and improved survival of patients with BRAF-V600E/K melanoma, but their effectiveness is limited by resistance. Beyond melanoma, current clinical RAF inhibitors show modest efficacy when used for colorectal and thyroid BRAF-V600E tumours or for tumours harbouring BRAF alterations other than the V600 mutation. Accumulated experimental and clinical evidence indicates that the complex biochemical mechanisms of RAF kinase signalling account both for the effectiveness of RAF inhibitors and for the various mechanisms of tumour resistance to them. Recently, a number of next-generation RAF inhibitors, with diverse structural and biochemical properties, have entered preclinical and clinical development. In this Review, we discuss the current understanding of RAF kinase regulation, mechanisms of inhibitor action and related clinical resistance to these drugs. The recent elucidation of critical structural and biochemical aspects of RAF inhibitor action, combined with the availability of a number of structurally diverse RAF inhibitors currently in preclinical and clinical development, will enable the design of more effective RAF inhibitors and RAF-inhibitor-based therapeutic strategies, tailored to different clinical contexts.

Activating MAPK1 (ERK2) mutation in an aggressive case of disseminated juvenile xanthogranuloma.

Juvenile xanthogranuloma (JXG) is a rare histiocytic disorder that is usually benign and self-limiting. We present a case of atypical, aggressive JXG harboring a novel mitogen-activated protein kinase (MAPK) pathway mutation in the MAPK1 gene, which encodes mitogen-activated protein kinase 1 or extracellular signal-regulated 2 (ERK2). Our analysis revealed that the mutation results in constitutive ERK activation that is resistant to BRAF or MEK inhibitors but susceptible to an ERK inhibitor. These data highlight the importance of identifying specific MAPK pathway alterations as part of the diagnostic workup for patients with histiocytic disorders rather than initiating empiric treatment with MEK inhibitors.