Oncogenic BRAF kinase deregulates the ERK signaling pathway in a large number of human tumors. FDA-approved BRAF inhibitors for BRAFV600E/K tumors have provided impressive clinical responses ...extending survival of melanoma patients. However, these drugs display paradoxical activation in normal tissue with BRAFWT due to RAF transactivation and priming, acquired drug resistance, and limited clinical effectiveness in non-V600 BRAF-dependent tumors, underscoring the urgent need to develop improved BRAF inhibitors. This review provides an overview of recent structural and biochemical insights into the mechanisms of BRAF regulation by BRAF inhibitors that are linked to their clinical activity, clinical liabilities, and medicinal chemistry properties. The effectiveness and challenges of structurally diverse next generation RAF inhibitors currently in preclinical and clinical development are discussed, along with mechanistic insights for developing more effective RAF inhibitors targeting different oncogenic BRAF conformations.
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.
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IJS, NUK, SBMB, UL, UM, UPUK
The BCL-2 family protein BAX has essential activity in mitochondrial regulation of cell death. While BAX activity ensures tissue homeostasis, when dysregulated it contributes to aberrant cell death ...in several diseases. During cellular stress BAX is transformed from an inactive cytosolic conformation to a toxic mitochondrial oligomer. Although the BAX transformation process is not well understood, drugs that interfere with this process are useful research tools and potential therapeutics. Here, we show that Eltrombopag, an FDA-approved drug, is a direct inhibitor of BAX. Eltrombopag binds the BAX trigger site distinctly from BAX activators, preventing them from triggering BAX conformational transformation and simultaneously promoting stabilization of the inactive BAX structure. Accordingly, Eltrombopag is capable of inhibiting BAX-mediated apoptosis induced by cytotoxic stimuli. Our data demonstrate structure-function insights into a mechanism of BAX inhibition and reveal a mechanism for Eltrombopag that may expand its use in diseases of uncontrolled cell death.
Mitofusins (MFNs) promote fusion-mediated mitochondrial content exchange and subcellular trafficking. Mutations in
cause neurodegenerative Charcot-Marie-Tooth disease type 2A (CMT2A). We showed that ...MFN2 activity can be determined by Met
and His
interactions with Asp
and Leu
and controlled by PINK1 kinase-mediated phosphorylation of adjacent MFN2 Ser
Small-molecule mimics of the peptide-peptide interface of MFN2 disrupted this interaction, allosterically activating MFN2 and promoting mitochondrial fusion. These first-in-class mitofusin agonists overcame dominant mitochondrial defects provoked in cultured neurons by CMT2A mutants MFN2 Arg
→Gln
and MFN2 Thr
→Met
, as demonstrated by amelioration of mitochondrial dysmotility, fragmentation, depolarization, and clumping. A mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of MFN2 Thr
→Met
mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism and/or trafficking.
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BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAF
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.
The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells
. This process requires major molecular adaptations to allow HSCs to meet ...the regulatory and metabolic requirements for cell division
. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)
, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
BAX is a critical effector of the mitochondrial cell death pathway in response to a diverse range of stimuli in physiological and disease contexts. Upon binding by BH3-only proteins, cytosolic BAX ...undergoes conformational activation and translocation, resulting in mitochondrial outer-membrane permeabilization. Efforts to rationally target BAX and develop inhibitors have been elusive, despite the clear therapeutic potential of inhibiting BAX-mediated cell death in a host of diseases. Here, we describe a class of small-molecule BAX inhibitors, termed BAIs, that bind directly to a previously unrecognized pocket and allosterically inhibit BAX activation. BAI binding around the hydrophobic helix α5 using hydrophobic and hydrogen bonding interactions stabilizes key areas of the hydrophobic core. BAIs inhibit conformational events in BAX activation that prevent BAX mitochondrial translocation and oligomerization. Our data highlight a novel paradigm for effective and selective pharmacological targeting of BAX to enable rational development of inhibitors of BAX-mediated cell death.
The BCL2 family plays important roles in acute myeloid leukemia (AML). Venetoclax, a selective BCL2 inhibitor, has received FDA approval for the treatment of AML. However, drug resistance ensues ...after prolonged treatment, highlighting the need for a greater understanding of the underlying mechanisms. Using a genome-wide CRISPR/Cas9 screen in human AML, we identified genes whose inactivation sensitizes AML blasts to venetoclax. Genes involved in mitochondrial organization and function were significantly depleted throughout our screen, including the mitochondrial chaperonin
. We demonstrated that CLPB is upregulated in human AML, it is further induced upon acquisition of venetoclax resistance, and its ablation sensitizes AML to venetoclax. Mechanistically, CLPB maintains the mitochondrial cristae structure via its interaction with the cristae-shaping protein OPA1, whereas its loss promotes apoptosis by inducing cristae remodeling and mitochondrial stress responses. Overall, our data suggest that targeting mitochondrial architecture may provide a promising approach to circumvent venetoclax resistance. SIGNIFICANCE: A genome-wide CRISPR/Cas9 screen reveals genes involved in mitochondrial biological processes participate in the acquisition of venetoclax resistance. Loss of the mitochondrial protein CLPB leads to structural and functional defects of mitochondria, hence sensitizing AML cells to apoptosis. Targeting CLPB synergizes with venetoclax and the venetoclax/azacitidine combination in AML in a p53-independent manner.
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Deregulation of the BCL-2 family interaction network ensures cancer resistance to apoptosis and is a major challenge to current treatments. Cancer cells commonly evade apoptosis through upregulation ...of the BCL-2 anti-apoptotic proteins; however, more resistant cancers also downregulate or inactivate pro-apoptotic proteins to suppress apoptosis. Here, we find that apoptosis resistance in a diverse panel of solid and hematological malignancies is mediated by both overexpression of BCL-XL and an unprimed apoptotic state, limiting direct and indirect activation mechanisms of pro-apoptotic BAX. Both survival mechanisms can be overcome by the combination of an orally bioavailable BAX activator, BTSA1.2 with Navitoclax. The combination demonstrates synergistic efficacy in apoptosis-resistant cancer cells, xenografts, and patient-derived tumors while sparing healthy tissues. Additionally, functional assays and genomic markers are identified to predict sensitive tumors to the combination treatment. These findings advance the understanding of apoptosis resistance mechanisms and demonstrate a novel therapeutic strategy for cancer treatment.
Chaperone-mediated autophagy (CMA) contributes to cellular quality control and the cellular response to stress through the selective degradation of cytosolic proteins in lysosomes. A decrease in CMA ...activity occurs in aging and in age-related disorders (for example, neurodegenerative diseases and diabetes). Although prevention of this age-dependent decline through genetic manipulation in mice has proven beneficial, chemical modulation of CMA is not currently possible, owing in part to the lack of information on the signaling mechanisms that modulate this pathway. In this work, we report that signaling through retinoic acid receptor α (RARα) inhibits CMA and apply structure-based chemical design to develop synthetic derivatives of all-trans-retinoic acid to specifically neutralize this inhibitory effect. We demonstrate that chemical enhancement of CMA protects cells from oxidative stress and from proteotoxicity, supporting a potential therapeutic opportunity when reduced CMA contributes to cellular dysfunction and disease.
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