Ben Powell

Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF -mutant melanoma

B-RAF is the most frequently mutated protein kinase in human cancers. The finding that oncogenic mutations in BRAF are common in melanoma, followed by the demonstration that these tumours are dependent on the RAF/MEK/ERK pathway, offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients. Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity. Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts. Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032 (ref. 5). In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment. This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumour regressions. At higher drug exposures afforded by a new amorphous drug formulation, greater than 80% inhibition of ERK phosphorylation in the tumours of patients correlated with clinical response. Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily. These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

BRAFV600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting “active” protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-RafV600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-RafV600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-RafV600E-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-RafV600E-positive cells. In B-RafV600E-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-RafV600E-driven tumors.

RAF inhibitors that evade paradoxical MAPK pathway activation

Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed ‘paradox breakers’) that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394.

Scaffold-based discovery of indeglitazar, a PPAR pan-active anti-diabetic agent

In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with high-throughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPARα, PPARγ, and PPARδ. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPARγ, to circumvent the clinically observed side effects of full PPARγ agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPARγ. Compared with full PPARγ-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).

Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor

Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.

BRD4 profiling identifies critical Chronic Lymphocytic Leukemia oncogenic circuits and reveals sensitivity to PLX51107, a novel structurally distinct BET inhibitor

Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is overexpressed in CLL and is enriched proximal to genes upregulated or de novo expressed in CLL with known functions in disease pathogenesis and progression. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel in vitro and in vivo pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in preclinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of BRD4 profiling in other cancers.Significance: To date, functional studies of BRD4 in CLL are lacking. Through integrated genomic, functional, and pharmacologic analyses, we uncover the existence of BRD4-regulated core CLL transcriptional programs and present preclinical proof-of-concept studies validating BET inhibition as an epigenetic approach to target BCR signaling in CLL. Cancer Discov; 8(4); 458-77. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.

Abstract 4711: Broad anti-tumor activity of a novel BET bromodomain inhibitor

Inhibitors against the bromodomain and extra terminal domain (BET) family of proteins have been pursued as promising oncology agents based on growing understanding of epigenetic control of disease processes. Through scaffold-based and crystallography-guided drug design, we discovered PLX51107, a potent and selective small molecule inhibitor of the BET family bromodomains. PLX51107 is structurally unrelated to the benzodiazepines such as JQ1, I-BET762, and OTX015 and other published BET inhibitors. PLX51107 exhibits low nanomolar potency in blocking interactions mediated by the four BET family proteins BRD2, BRD3, BRD4, and BRDT. Pharmacologic inhibition of BET proteins by PLX51107 suppresses the transcription of genes essential for tumor growth and survival and leads to selective killing of cancer cell lines across a broad range of hematologic malignancies (e.g. leukemia, lymphoma and multiple myeloma). A subset of solid tumors (e.g. melanoma and SCLC) is also sensitive to growth inhibition by the BET inhibitor PLX51107. Novel biomarkers in these diseases have been identified. PLX51107 is well tolerated and has sufficient potency and oral bioavailability to demonstrate in vivo efficacy in animal models of a variety of tumor types, representing both hematologic and solid tumors of diverse genetic backgrounds. In combination studies, PLX51107 showed potential to improve the efficacy (response rates and duration of response) of other anticancer treatments without increased toxicity. These results support further development of PLX51107 as an epigenetic-based therapy for a variety of cancer indications. Citation Format: Yan Ma, Ben Powell, Jiazhong Zhang, Ying Zhang, Heidi Carias, Ullrich Schwertschlag, Gaston Habets, Prabha Ibrahim, Wayne Spevak, Chao Zhang, Gideon Bollag. Broad anti-tumor activity of a novel BET bromodomain inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4711.

Abstract 553: Discovery of novel TRK/FMS dual inhibitors as therapeutic candidates for pancreatic cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Pancreatic cancer is a deadly malignancy in need of effective treatments. The most common driving oncogenes in pancreatic cancer – KRAS, p53, SMAD4, CDKN2A and CTNNB1 – historically have been difficult drug targets to modulate pharmacologically. An alternative approach could be to target regulators of tumor-stroma interactions. Pancreatic cancer invasion into neural tissue precedes tumor expansion. This perineural invasion is strongly associated with neural hypertrophy, pain and poor survival, and neurotrophins and their receptors (TRKs) are key suspects in mediating this process. At the same time, infiltrating macrophages are an additional component of the tumor microenvironment that supports tumor growth, invasion and inflammation, and the receptor for CSF-1 (FMS) is a key mediator of the function and survival of these macrophages. Both TRKs and FMS are transmembrane proteins with tyrosine kinase activities that can be inhibited with small molecule agents. We have developed a series of TRK/FMS dual inhibitors to target cancers that exhibit both neural and inflammatory components. These compounds inhibit biochemical TRK and FMS kinases with IC50 80% at doses of 20 mg/kg qd, and without body weight changes, showing that efficacy is not due to nonspecific toxicity. In a mouse Complete Freunds Adjuvant (CFA) model, robust efficacy was demonstrated in readouts of paw edema, thermal hyperalgesia and mechanical allodynia, showing that these compounds reduce swelling and pain. In the TRK-driven SK-N-SH xenograft model, tumor growth reduction of >50% was observed. These compounds also showed anti-tumor efficacy of >40% in an orthotopic Panc-1 (pancreatic cancer) model. Evaluation of compound effects on perineural invasion and inflammation is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 553. doi:10.1158/1538-7445.AM2011-553