Search

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.

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 G

Supplementary Figures and Legends

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

Supplementary Video 2

Supplementary Tables

Supplementary Video 5

Supplementary Video 4

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

Supplementary Video 1

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

Supplementary Video 6

Supplementary Methods, Figures 1-5 from mTOR Kinase Inhibition Causes Feedback-Dependent Biphasic Regulation of AKT Signaling

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

Supplementary Video 3

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

Purpose: To test second-line personalized medicine combination therapies, based on genomic and proteomic data, in patient-derived xenograft (PDX) models.Experimental Design: We established 12 PDXs from BRAF inhibitor–progressed melanoma patients. Following expansion, PDXs were analyzed using targeted sequencing and reverse-phase protein arrays. By using multi-arm preclinical trial designs, we identified efficacious precision medicine approaches.Results: We identified alterations previously described as drivers of resistance: NRAS mutations in 3 PDXs, MAP2K1 (MEK1) mutations in 2, BRAF amplification in 4, and aberrant PTEN in 7. At the protein level, re-activation of phospho-MAPK predominated, with parallel activation of PI3K in a subset. Second-line efficacy of the pan-PI3K inhibitor BKM120 with either BRAF (encorafenib)/MEK (binimetinib) inhibitor combination or the ERK inhibitor VX-11e was confirmed in vivo. Amplification of MET was observed in 3 PDX models, a higher frequency than expected and a possible novel mechanism of resistance. Importantly, MET amplification alone did not predict sensitivity to the MET inhibitor capmatinib. In contrast, capmatinib as single agent resulted in significant but transient tumor regression in a PDX with resistance to BRAF/MEK combination therapy and high pMET. The triple combination capmatinib/encorafenib/binimetinib resulted in complete and sustained tumor regression in all animals.Conclusions: Genomic and proteomic data integration identifies dual-core pathway inhibition as well as MET as combinatorial targets. These studies provide evidence for biomarker development to appropriately select personalized therapies of patients and avoid treatment failures. Clin Cancer Res; 22(7); 1592–602. ©2015 AACR.See related commentary by Hartsough and Aplin, p. 1550

A. PCA of protein expression profiles of WM3965 control and progression tumor grafts. B. unsupervised hierarchical clustering of proteins identified in the PCA.

Supplementary Table S1: Summary of melanoma patients with clinical follow-up. Figure Legends

Tumors with mutant RAS are often dependent on extracellular signal–regulated kinase (ERK) signaling for growth; however, MEK inhibitors have only marginal antitumor activity in these tumors. MEK inhibitors relieve ERK-dependent feedback inhibition of RAF and cause induction of MEK phosphorylation. We have now identified a MEK inhibitor, CH5126766 (RO5126766), that has the unique property of inhibiting RAF kinase as well. CH5126766 binding causes MEK to adopt a conformation in which it cannot be phosphorylated by and released from RAF. This results in formation of a stable MEK/RAF complex and inhibition of RAF kinase. Consistent with this mechanism, this drug does not induce MEK phosphorylation. CH5126766 inhibits ERK signaling output more effectively than a standard MEK inhibitor that induces MEK phosphorylation and has potent antitumor activity as well. These results suggest that relief of RAF feedback limits pathway inhibition by standard MEK inhibitors. CH5126766 represents a new type of MEK inhibitor that causes MEK to become a dominant-negative inhibitor of RAF and that, in doing so, may have enhanced therapeutic activity in ERK-dependent tumors with mutant RAS. Cancer Res; 73(13); 4050–60. ©2013 AACR.

Supplementary Figure S1 - PDF file 70K, Induction of p27Kip1 protein and cell cycle arrest by CH5126766

Supplementary Materials and Methods - PDF file 65K, Antibodies and kinase panel assays

Supplementary Table S4 - PDF file 56K, Binding constant between MEK1 and BRAF or CRAF, related to Figure 3. His6-MEK1 binding to immobilized GST-BRAF or GST-CRAF in the presence of 500 �M ATP, 500 �M ATP plus 3 �M CH5126766 or 500 �M ATP plus 3 �M PD0325901 were analyzed with SPR

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.

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

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.

Cyclin-dependent kinases (CDKs) 4 and 6 inhibitors (CDK4/6is) 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 CDK4/6is. In contrast, tumor cells expressing both CDK4 and CDK6 have increased reliance on CDK6 to ensure cell cycle progression. We discovered that CDK4/6is 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, CDK4/6is 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 CDK4/6is and CDK4/6i-derived degraders and the need for new inhibitors targeting the CDK4/6i-resistant, thermostable form of CDK6 for application as cancer therapeutics. Poulikakos and colleagues demonstrate that thermostable CDK6 complexes promote resistance to therapeutic targeting of CDK4/6 in cancer.

Oncogenic alterations of the RAS/RAF/MEK/ERK signaling pathway drives growth of over 40% of human tumors, frequently due to activating mutations of components of the pathway, such as RAS or BRAF. Targeting the pathway using BRAF inhibitors (BRAFi) in combination with MEK inhibitors (MEKi) is now the standard of care for metastatic melanoma patients harboring the BRAF(V600E) mutation. However, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including those with RAS activating mutations. The effectiveness of current clinical BRAFi relies on the fact that they bind selectively and inhibit the mutationally activated monomeric form of the BRAF(V600E) kinase in tumors but not wild-type BRAF in normal tissue where BRAF signals in its dimeric form. Yet, the development of adaptive resistance, frequently due to RAF dimerization, limits BRAFi therapeutic effectiveness. Another class of BRAFi equipotent for both monomeric and dimeric BRAF have been developed, but are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues as well. Recently, we identified and characterized a novel class of BRAFi that preferentially binds and inhibits dimeric BRAF over monomeric BRAF(V600E) (i.e. RAF dimer-selective inhibitors). Furthermore, we rationally designed a combinatorial approach utilizing a BRAF monomer-selective plus a BRAF dimer-selective inhibitor plus a MEK inhibitor, that potently disrupts the BRAF/MEK complex, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index. This triple combination potently suppressed tumor growth of multiple BRAF(V600E) colorectal cancer and melanoma models resistant to the current clinical BRAFi and MEKi combinations both in vitro and in vivo. To design effective therapeutic approaches for RAS-mutated tumors, we also assessed the effectiveness of RAF and MEK inhibitors belonging to diverse biochemical and structural classes, either alone or in combination in RAS-mutant tumor models and normal cells with wild-type RAS signaling. We found that RAF dimer-selective inhibitors potently antagonized MAPK signaling and growth in various RAS mutant cancer models as compared to normal cells. We found further that RAF and MEK inhibitor combinations belonging to certain biochemical classes resulted in more potent MAPK suppression in RAS-mutant tumor as compared to wild-type RAS normal cells (“therapeutic synergy”) and should be prioritized for clinical testing. In contrast, those combinations which are more potent in normal over tumor cells (“adverse synergy”) are unlikely to be clinically successful. Thus, rationally designed combinations of selected RAFi and MEKi, based on their distinct biochemical properties, can uncover new therapeutic opportunities tailored for more effective targeting of BRAF or RAS mutant tumors. Citation Format: Ana Orive-Ramos, Christos Adamopoulos, Beau Baars, Jason Lam, Ankita Punetha, Jason Huang, Vasileios I. Petrou, Stuart A. Aaronson, Poulikos I. Poulikakos. Designing RAF and MEK inhibitor combinations based on their biochemical properties to effectively target MAPK-driven cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5734.

Activating RAS mutations constitute the majority of recurrent mutations found in MAPK-driven human tumors. However, RAS mutants have long been considered undruggable, and therefore efforts have been predominantly focused on targeting downstream MAPK signaling. We and others have shown that, MAPK inhibition using MEK inhibitors (MEKi) have limited effectiveness in such tumors due to adaptive resistance. A major mechanism of adaptive resistance involves relief of negative feedback that promotes reactivation of RTK signaling, which in turn activates RAS and results in MAPK rebound. The recent development of direct (i.e. RAS(G12C)) and indirect (i.e. SHP2i, SOSi) inhibitors of RAS have uncovered the dependence of activity of RAS(G12C) and of other RAS mutants on upstream signaling. We and others have previously characterized sensitivity of various RAS mutants in cancer to either SHP2 inhibition (SHP2i), or to combination of SHP2i with MEKi, but a systematic characterization of most RAS mutants commonly expressed in cancer has been lacking. In this study, by combining biochemical and chemical genetic approaches we identified two classes of RAS-mutants, based on their degree of dependency on RTK/SHP2 signaling and adaptation to MEK inhibition. One class of RAS mutants, including most RASQ61X, show complete independence from RTK/SHP2 signaling and are fully resistant to either SHP2i alone or in combination with MEKi. The other class includes the majority of RAS mutants and is characterized by relative resistance to SHP2i, or the combination of SHP2i plus MEKi, compared to cells expressing wild-type RAS, including normal cells. Early clinical data using single agent SHP2i or combinations of SHP2i with MEKi for the treatment of patients with RAS-mutant tumors suggested modest activity as well as dose-limiting on-target (i.e. MAPK pathway related) toxicities. Our findings predict that achieving an acceptable therapeutic index by either approach for treatment of RAS-mutant tumors will be challenging, and they support instead SHP2i-based therapeutic efforts in patients with MAPK-driven tumors expressing wild-type RAS, or in combination with RAS-mutant selective inhibitors. Collectively this work provides a framework for the design of SHP2i-based treatments for patients with MAPK-driven tumors, the majority of which currently have no effective targeted therapeutic options. Citation Format: Beau Baars, Christos Adamopoulos, Ana Orive-Ramos, Stuart A. Aaronson, Poulikos I. Poulikakos. RTK/SHP2 dependency and feedback adaptation to MEK inhibition defines sensitivity of RAS-mutant proteins to SHP2 inhibitor-based therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3413.

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

PI3K/AKT/mTOR signaling pathway is one of the most frequently dysregulated pathway in cancer development and the serine/threonine kinase AKT functions as the key node in this pathway to regulate multiple cellular and physiological processes. AKT inhibitors that competitively bind the ATP pocket have been evaluated in clinical trials and have a tolerable toxicity profile with greater efficacy for tumors with pathway mutations; however, many PI3K/AKT pathway mutant tumors remain resistant. Proteolysis targeting chimeras (PROTACs) are a powerful targeted protein degradation technology that hijacks the cellular ubiquitin-proteasome system to induce selective polyubiquitination and degradation of the target proteins. Using this technology, we designed and developed a library of novel small-molecules putative degraders to degrade AKT by recruiting either the cereblon (CRBN) or von Hippel-Lindau (VHL) E3 ligase, that have the ability to degrade AKT in cells and lower downstream signaling to various extents. Though extensive structure-activity relationship studies on various linkers, E3 ligase ligands, and AKT binding moieties, we identified a novel VHL-recruiting AKT degrader, MS21, and characterized it using a panel of 38 cancer cell lines with diverse genotypes and tissue origins. Our results suggest that efficient pharmacologic degradation of AKT phosphorylated on threonine 308 and serine 473 leads to selective inhibition of the growth of tumor cells with alterations of HER2, PIK3CA, PTEN, or AKT1. In these PI3K/PTEN pathway mutant lines, AKT degradation by MS21 was superior to AKT kinase inhibition for reducing cell growth and sustaining lower signaling over many days, and inhibited tumor cell growths through lowering the level of Aurora Kinase B, which we found to be an AKT substrate protein. 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 its degradation rescued cells from growth inhibition by MS21. In addition, resistance to MS21 was found to be associated with low levels of baseline AKT phosphorylation in cells as well as mutation of either KRAS or BRAF, and resistance to MS21 could be overcome by the combination treatment of a MEK inhibitor trametinib with MS21, which not only inhibited MEK but also increased AKT phosphorylation and enhanced AKT degradation. Pan-cancer analysis identified that 19% of cases have PI3K/PTEN pathway mutation without RAS pathway mutation, suggesting that these cancer patients could benefit from AKT degrader therapy that leads to loss of AURKB. Citation Format: Jia Xu, Xufen Yu, Tiphaine C. Martin, Ankita Bansal, Kakit Cheung, Abigail Lubin, Elias Stratikopoulos, Kaitlyn M. Cahuzac, Li Wang, Ling Xie, Royce Zhou, Yudao Shen, Xuewei Wu, Shen Yao, Ruifang Qiao, Poulikos I. Poulikakos, Xian Chen, Jing Liu, Jian Jin, Ramon Parsons. AKT degradation selectively inhibits the growth of PI3K/PTEN pathway mutant cancers with wild type KRAS and BRAF by destabilizing Aurora kinase B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB195.

BRAF kinase, a critical component of the RAS/MAPK signaling pathway, requires dimerization for its catalytic activity, whereas the oncoprotein BRAF(V600E) can be catalytically active as a monomer. Current clinical BRAF inhibitors selectively bind and inhibit monomeric over dimeric BRAF (i.e. “BRAF monomer-selective”) providing a high therapeutic index and are now standard practice in combination with MEK inhibitors in the treatment of patients harboring BRAF(V600E) tumors. However adaptive resistance due to RAF dimerization limits their effectiveness. Recently, RAF inhibitors that equipotently inhibit both monomeric and dimeric BRAF have been developed, but they are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues. We identified and characterized a novel class of BRAF inhibitors that preferentially bind and inhibit dimeric over monomeric BRAF(V600E) (i.e. “BRAF dimer-selective inhibitors”). Biochemical analysis using BRAF dimer-selective inhibitors revealed that the two forms of dimeric and monomeric BRAF(V600E) differ in their conformation in their active site and their strength of interaction with MEK. Further, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index, we assessed the triple combination of BRAF monomer- plus BRAF dimer selective inhibitor plus a MEK inhibitor that disrupts the BRAF-MEK complex. The triple combination potently suppressed tumor growth in multiple colorectal and melanoma BRAF(V600E) cell line- and in vivo models that were resistant to the current clinical BRAF and MEK inhibitor combination. Strikingly, while the double combination treatment with the BRAF dimer-selective inhibitor and MEK inhibitor was associated with a significant gradual weight loss in vivo, the triple combination showed no weight loss or other apparent toxicities, indicating a higher Therapeutic Index. Finally, off-label use of the triple combination in patients with advanced BRAF(V600E) cancers that progressed on standard therapies achieved durable tumor control with minimal toxicities. Thus, a rationally designed combinatorial approach of conformation-selective BRAF and MEK inhibitors may be a highly effective and well tolerated therapeutic strategy for patients with BRAF(V600E) tumors. Overall, our work supports the notion that for most tumors a “3-drug” combinatorial strategy will potentially be most effective: one drug targeting MAPK signaling directly, such as a MEK inhibitor (“pathway” inhibitor), one drug targeting mutated BRAF or RAS selectively in the tumor (“therapeutic index” inhibitor, e.g. BRAF monomer-selective inhibitor, RAS(G12C) inhibitors, etc) and one drug targeting components of the feedback loop that is responsible for adaptive resistance (“feedback” inhibitor, e.g. BRAF dimer-selective inhibitors, SHP2 or SOS inhibitors, etc). Our data contribute to the development of a roadmap for the treatment of MAPK-driven tumors using drug combinations tailored to the specific driver oncoprotein. Citation Format: Christos Adamopoulos, Tamer Ahmed, Peter M. Ung, Min Xiao, Stuart Aaronson, Celina Ang, Vito Rebecca, Avner Schlessinger, Meenhard Herlyn, Poulikos I. Poulikakos. BRAF dimer-selective inhibitors synergize with BRAF monomer-selective inhibitors to overcome adaptive resistance and retain therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB083.

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., FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which limits their clinical efficacy in tumors expressing BRAFV600E mutant monomers. Here the authors identify FDA-approved Ponatinib as an effective inhibitor of BRAF monomers and dimers and designed PHI1, an inhibitor with a unique mode of action and selectivity for oncogenic BRAF dimers.

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.

Hogstad et al. show that the somatic BRAFV600E mutation in myeloid dendritic cell precursors in Langerhans cell histiocytosis promotes lesion formation through impaired dendritic cell migration and resistance to apoptosis, which can be rescued with targeted MAPK pathway inhibition., Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with constitutively activated mitogen-activated protein kinase (MAPK) pathway signaling. Approximately 60% of LCH patients harbor somatic BRAFV600E mutations localizing to CD207+ DCs within lesions. However, the mechanisms driving BRAFV600E+ LCH cell accumulation in lesions remain unknown. Here we show that sustained extracellular signal–related kinase activity induced by BRAFV600E inhibits C-C motif chemokine receptor 7 (CCR7)–mediated DC migration, trapping DCs in tissue lesions. Additionally, BRAFV600E increases expression of BCL2-like protein 1 (BCL2L1) in DCs, resulting in resistance to apoptosis. Pharmacological MAPK inhibition restores migration and apoptosis potential in a mouse LCH model, as well as in primary human LCH cells. We also demonstrate that MEK inhibitor-loaded nanoparticles have the capacity to concentrate drug delivery to phagocytic cells, significantly reducing off-target toxicity. Collectively, our results indicate that MAPK tightly suppresses DC migration and augments DC survival, rendering DCs in LCH lesions trapped and resistant to cell death.

Background: Multiple Myeloma (MM) is a progressive plasma cell neoplasm characterized by heterogeneous clonal expansion. Despite numerous targeted therapy options, failing anti-BCMA CAR-T cell therapy represents a challenging treatment course with dismal prognosis. 53% of relapsed/refractory (rr) MM patients have shown emerging clones harboring mutations within the MAPK signaling pathway including the targetable BRAF V600E in 7% of cases. Allosteric kinase inhibition of MAPK pathway with BRAF monomer selective inhibitors +/- MEK inhibitor have been frequently attempted with rrMM patients with equivocal outcome on both disease progression and survival. This could be ascribed to the feedback activation of the pathway via BRAF dimers induction. In solid tumor contexts, a recent study has revealed that the multi-kinase inhibitor regorafenib is a potent and selective inhibitor of dimeric BRAF. Moreover, the study demonstrated overcoming of this negative feedback by combining two inhibitors targeting BRAF in its monomeric and its dimeric form, respectively, with MEK inhibition leading to more efficacious and tolerable treatment (Adamopoulos C. et al. Cancer Discov. 2021). Here, we show the applicability, effectiveness, and tolerability of the aforementioned triple therapy strategy in hematological malignancy context to salvage post BCMA CAR-T cell therapy in a BRAF V600E rrMM patient. Material and Methods: CD138 + selected cells were collected from bone marrow (BM) and patient's plasmacytoma before and after CAR-T cell therapy relapse, respectively. Samples were processed using the standard GATK 4 workflows and a consensus mutation calling approach was used to call somatic mutations using a matched normal obtained from peripheral blood. PhyloWGS (Deshwar et al, Genome Biol 2015) was then used to perform intra-tumor heterogeneity modeling and phylogeny estimation. CD138 + selected cells from BM of rrMM patient were treated with encorafenib (50nM) and binimetinib (250 nM) +/- regorafinib (1mM) for 48 hours, cells were harvested, lysed and immunoblotted with BRAF V600E and pERK antibodies. In parallel, rrMM patient BM selected CD138 + cells' sensitivity to MAPK inhibitors were assessed by Travera. Cells are incubated with different inhibitors at varying concentrations and combinations for 15 hours before having their single-cell mass distributions measured. Sensitivity is then characterized by the relative statistical difference (Hellinger distance) between conditions and untreated controls. A rrMM patient was started on targeted triple therapy using the following combination: BRAF monomer-inhibitor, dabrafenib (100mg, orally twice daily), a MEK-inhibitor, trametinib (1.5mg, orally for 21/28 days daily), and a BRAF dimer inhibitor, regorafenib (40mg, orally once daily). Results: Whole Exome Sequencing (WES) showed a BRAF V600E mutation was present prior to CAR-T cell therapy and persisted through subsequent treatments. The variant allele frequency of the mutation was 41% post CAR-T cell therapy (Figure 1A). Western blot of CD138 + cells confirmed BRAF V600E mutation with a specific antibody, and showed ERK phosphorylation, which was inhibited with trametinib. Longer exposure to trametinib confirmed a feedback loop activating MAPK which was abrogated with the BRAF dimer inhibitor regorafenib (Figure 1B). Travera analysis showed CD138 + cells' sensitivity to the combination of trametinib at 5mM, and dabrafenib at 1µM. Adding regorafenib at 1µM could slightly improve the response compared to the double combination. (Figure 1C). Clinically, three months of the triple therapy administration has led to prompt reduction in subcutaneous skin lesions as well as 80% reduction in free lambda chain (Figure 1D). Furthermore, the patient exhibited good tolerance to all three medications with minimal side effects (grade 1 fatigue). The treatment regimen allowed the patient to carry out activities of daily living and resume work. Conclusion: We have shown the applicability, effectiveness, and tolerability of the triple MAPK inhibition in a hematological malignancy context to salvage post BCMA CAR-T cell therapy in a rrMM patient with BRAF V600E mutation. Also, we have revealed the power of routine WES in tracking clonal evolution, and identifying targetable mutations which allows tailoring of therapeutics for MM patients. Figure 1 Figure 1. Disclosures Stevens: Travera: Current Employment. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

Background Despite a growing arsenal of treatment choices, patient relapse post-BCMA-targeted CART therapy remains a challenge and the therapeutic path is still undefined. Among the most frequently observed actionable mutations, BRAF V600E, is present in ~7% of multiple myeloma (MM) patients. The availability of selective inhibitors of BRAF V600E makes this a valuable therapeutic target. However, the clinical efficacy of these inhibitors has been limited and short-lived, frequently due to feedback-induction of BRAF dimers. Novel studies revealed that the multi-kinase inhibitor regorafenib is a potent and selective inhibitor of dimeric BRAF. They have further demonstrated relief of these negative outcomes by inhibiting BRAF in both its monomeric and dimeric form in combination with MEK inhibition leading to more efficacious and tolerable treatment. (Adamopoulos C. et al. Cancer Discov. 21). We present a case of a MM patient with BRAF V600E mutation salvaged with a triple therapy inhibition strategy after relapsing following anti-BCMA CART therapy. Method A 61-year-old male with penta-refractory MM (IgA lambda), ISS stage 3 with hyperdiploidy, gain of 1q21 and del13 was treated with anti-BCMA CART therapy, achieving a best response of VGPR, and disease progression after 6 months. He was temporarily salvaged with BCNU/Mel ASCT and achieved a best response of PR until progression with extramedullary disease (subcutaneous skin lesions in lower extremities) and elevated lambda free light chains (FLC, 126.4mg/l) at 6 months. Results Targeted sequencing showed a BRAF V600E mutation was present prior to CART therapy in his bone marrow and persisted in his bone marrow and a plasmacytoma with a VAF of 41% at this current relapse. A western blot analysis of bone marrow aspirate confirmed BRAF V600E with a specific antibody, and showed phosphorylation of ERK, which was diminished with trametinib. Longer exposure to trametinib confirmed a feedback loop activating MAPK which was abrogated with the BRAF dimer inhibitor regorafenib. With these findings the patient was started on targeted therapy based on combination of a BRAF monomer-inhibitor, dabrafenib (100mg, orally twice daily), a MEK-inhibitor, trametinib (1.5mg, orally for 21/28 days daily), and a BRAF dimer inhibitor, regorafenib (40mg, orally once daily). Within 3 months of treatment initiation, prompt reduction in subcutaneous skin lesions and 80% reduction in free lambda FLC (27.5 mg/l) was observed. Furthermore, the patient had good tolerance to all three medications with minimal side effects (grade 1 fatigue) and continues treatment. The treatment regimen allowed the patient to carry out activities of daily living and return to work. Conclusion Post-CART failure treatment is a challenging and unmet need. NGS may identify targeted therapy that would be able to salvage patients with a tolerable side effect profile.

BRAF kinase, a critical component of the RAS/MAPK signaling pathway, requires dimerization for its catalytic activity, whereas the oncoprotein BRAF(V600E) can be catalytically active as a monomer. Current clinical RAF inhibitors selectively bind and inhibit monomeric over dimeric BRAF providing a high therapeutic index and are now standard practice in combination with MEK inhibitors in the treatment of patients harboring BRAF(V600E) tumors. However adaptive resistance due to RAF dimerization limits their effectiveness. Recently, RAF inhibitors that equipotently inhibit both monomeric and dimeric RAF have been developed, but they are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues. Here we identify and characterize a third class of RAF inhibitors that preferentially binds and inhibits dimeric over monomeric BRAF(V600E). Using long-timescale molecular dynamics (MD) simulations, we observed distinct dynamic features of RAF inhibitors bound to monomeric and dimeric BRAF, thus providing a structural explanation for the selectivity of these drugs. Biochemical analysis using RAF dimer selective inhibitors revealed that the two forms of dimeric and monomeric BRAF(V600E) differ in their conformation in their active site and their strength of interaction with MEK. Further, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index, we assessed the triple combination of RAF monomer- plus RAF dimer selective inhibitor plus a MEK inhibitor that potently disrupts the BRAF-MEK complex. The triple combination potently suppressed tumor growth in multiple colorectal and melanoma BRAF(V600E) cell line- and in vivo models that were resistant to the current clinical RAF and MEK inhibitor combination. Strikingly, while the double combination treatment with the RAF dimer-selective inhibitor and MEK inhibitor was associated with a significant gradual weight loss in vivo, the triple combination showed no weight loss or other apparent toxicities, indicating a higher therapeutic index. Finally, off-label use of the triple combination in a patient with a stage IV BRAF(V600E) colorectal cancer that progressed on standard therapies achieved durable tumor control with minimal toxicities. Thus, a rationally designed combinatorial approach of conformation-selective RAF and MEK inhibitors may be a highly effective and well tolerated therapeutic strategy for patients with BRAF(V600E) tumors. Citation Format: Christos Adamopoulos, Tamer A. Ahmed, Maxwell R. Tucker, Peter M. Ung, Min Xiao, Zoi Karoulia, Xuewei Wu, Stuart A. Aaronson, Celina Ang, Vito Rebecca, Avner Schlessinger, Meenhard Herlyn, Qi Wang, David E. Shaw, Poulikos I. Poulikakos. A strategy for effective targeting of oncogenic BRAF signaling with increased therapeutic index using conformation-selective RAF and MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 78.

CDK4 and CDK6 kinases are master regulators of cell cycle progression, and they have thus been attractive targets for cancer therapy. CDK4/6 inhibitors (CDK4/6i) showed significant clinical effectiveness in ER+ breast cancers, and three CDK4/6i are now FDA-approved for this indication. However, in several tumor types, CDK4/6i have only been modestly effective, but the underlying mechanism(s) accounting for the discrepancy have remained elusive. To identify the basis of resistance to CDK4/6i, we employed a variety of biochemical, genetic and proteomic approaches, we developed a potent and selective CDK4/6-directed degrader (PROTAC) to investigate in-cell binding of compounds and validated our findings in large tumor and clinical-based data. Our studies revealed that tumor response to CDK4/6i is critically determined by the expression of CDK6. Tumors with low CDK6 expression (CDK6-low) depend on CDK4, and are uniformly sensitive to CDK4/6i. CDK6-low tumors include both entire cancer types (e.g. Ewing Sarcomas, MCL), as well as subgroups of large tumor types, such as portion of non-small cell lung adenocarcinomas (NSCLC). We further validated this finding in NSCLC patients previously treated with CDK4/6i in a clinical trial. In contrast, tumors that express both CDK4 and CDK6 universally depend on CDK6, which is expressed as either CDK4/6i-sensitive CDK6 (CDK6-S) or CDK4/6-resistant CDK6 (CDK6-R) in different cells. Using a CDK4/6-directed PROTAC, we further found that CDK4/6i binds strongly CDK6-S but weakly CDK6-R, indicating different conformations adopted by CDK6 in the two states. An unbiased proteomic analysis identified binding of components of the HSP90/CDC37 complex associated with the state of CDK6: in tumors in which CDK6 is expressed as a thermo-unstable, strong HSP90/CDC37-client, CDK4/6i (and consequently CDK4/6 PROTACs) bind and potently inhibit CDK6 and downstream Rb/E2F signaling. In contrast, tumors resistant to CDK4/6i express CDK6 as a thermostable, weak HSP90/CDC37-client that binds weakly to CDK4/6i. Our data uncover the expression state of CDK6 and its dependence on the HSP90/CDC37 complex as a critical determinant of tumor response to CDK4/6i. We further identify low CDK6 expression as a molecular predictor of tumor sensitivity to current CDK4/6i, indicating a potential biomarker for selection of patients that are more likely to benefit from these drugs. Finally, our findings underline the need for novel inhibitors targeting the thermostable CDK6-R to be used for the treatment of the large number of patients with of Rb-proficient solid tumors that are resistant to current clinical CDK4/6i. Thus, this unexpected dualism in the dependence of CDK6 kinase on HSP90 explains resistance of a large portion of tumors to current CDK4/6i, and provides a roadmap for developing more effective CDK4/6-directed pharmacologic strategies for cancer therapy. Citation Format: Xuewei Wu, Xiaobao Yang, Yan Xiong, Ruitong Li, Takahiro Ito, Tamer Ahmed, Zoi Karoulia, Christos Adamopoulos, Hong Wang, Li Wang, Ling Xie, Beatrix Ueberheide, Stuart Aaronson, Xian Chen, William Sellers, Sean Buchanan, Jian Jin, Poulikos I. Poulikakos. Tumor resistance to CDK4/6 inhibitors and degraders determined by the expression state of CDK6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 41.

// Rikhia Chakraborty 1, 2 , Oliver A. Hampton 3, 5 , Harshal Abhyankar 1, 2 , Daniel J. Zinn 1, 2 , Amanda Grimes 1, 2 , Brooks Skull 1, 2 , Olive Eckstein 1, 2 , Nadia Mahmood 6 , David A. Wheeler 3, 5 , Dolores Lopez-Terrada 1, 4 , Tricia L. Peters 4 , John M. Hicks 4 , Tarek Elghetany 4 , Robert Krance 1, 2, 7 , Poulikos I. Poulikakos 8, 9, 10 , Miriam Merad 8, 9, 11 , Kenneth L. McClain 1, 2 , Carl E. Allen 1, 2 and Donald W. Parsons 1, 2, 3, 4, 5 1 Texas Children’s Cancer Center, Texas Children’s Hospital, Houston, TX 77030, USA 2 Department of Pediatrics, Division of Pediatric Hematology-Oncology, Baylor College of Medicine, Houston, TX 77030, USA 3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA 4 Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA 5 Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA 6 Body and Nuclear Radiology Sections, Texas Children’s Hospital, Houston, TX 77030, USA 7 Center for Cell and Gene Therapy, Houston, TX 77030, USA 8 Department of Oncological Sciences, Icahn School of Medicine, New York, NY 10029, USA 9 Tisch Cancer Institute, Icahn School of Medicine, New York, NY 10029, USA 10 Immunology Institute, Icahn School of Medicine, New York, NY 10029, USA 11 Department of Dermatology, Icahn School of Medicine, New York, NY 10029, USA Correspondence to: Donald W. Parsons, email: dwparson@txch.org Carl E. Allen, email: ceallen@txch.org Keywords: juvenile xanthogranuloma, MAPK1 , ERK activation, histiocytic disorder, somatic mutation Received: October 13, 2016 Accepted: March 13, 2017 Published: April 29, 2017 ABSTRACT 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.

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 BRAF(V600E). We recently discovered that the BRAF(V600E) mutation can also affect multipotent hematopoietic progenitor cells (HPCs) in multisystem LCH disease. How the BRAF(V600E) mutation in HPCs leads to LCH is not known. Here we show that enforced expression of the BRAF(V600E) 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.

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.

Summary The complex biochemical effects of RAF inhibitors account for both the effectiveness and mechanisms of resistance to these drugs, but a unified mechanistic model has been lacking. Here we show that RAF inhibitors exert their effects via two distinct allosteric mechanisms. Drug resistance due to dimerization is determined by the position of the αC helix stabilized by inhibitor, whereas inhibitor-induced RAF priming and dimerization are the result of inhibitor-induced formation of the RAF/RAS-GTP complex. The biochemical effect of RAF inhibitor in cells is the combined outcome of the two mechanisms. Therapeutic strategies including αC-helix-IN inhibitors are more effective in multiple mutant BRAF-driven tumor models, including colorectal and thyroid BRAF V600E cancers, in which first-generation RAF inhibitors have been ineffective.