Peter Hirth

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.

Vemurafenib: the first drug approved for BRAF -mutant cancer

The identification of driver oncogenes has provided important targets for drugs that can change the landscape of cancer therapies. One such example is the BRAF oncogene, which is found in about half of all melanomas as well as several other cancers. As a druggable kinase, oncogenic BRAF has become a crucial target of small-molecule drug discovery efforts. Following a rapid clinical development path, vemurafenib (Zelboraf; Plexxikon/Roche) was approved for the treatment of BRAF-mutated metastatic melanoma in the United States in August 2011 and the European Union in February 2012. This Review describes the underlying biology of BRAF, the technology used to identify vemurafenib and its clinical development milestones, along with future prospects based on lessons learned during its development.

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.

Structure-Guided Blockade of CSF1R Kinase in Tenosynovial Giant-Cell Tumor

BACKGROUND Expression of the colony-stimulating factor 1 (CSF1) gene is elevated in most tenosynovial giant-cell tumors. This observation has led to the discovery and clinical development of therapy targeting the CSF1 receptor (CSF1R). METHODS Using x-ray co-crystallography to guide our drug-discovery research, we generated a potent, selective CSF1R inhibitor, PLX3397, that traps the kinase in the autoinhibited conformation. We then conducted a multicenter, phase 1 trial in two parts to analyze this compound. In the first part, we evaluated escalations in the dose of PLX3397 that was administered orally in patients with solid tumors (dose-escalation study). In the second part, we evaluated PLX3397 at the chosen phase 2 dose in an extension cohort of patients with tenosynovial giant-cell tumors (extension study). Pharmacokinetic and tumor responses in the enrolled patients were assessed, and CSF1 in situ hybridization was performed to confirm the mechanism of action of PLX3397 and that the pattern of CSF1 expression was consistent with the pathological features of tenosynovial giant-cell tumor. RESULTS A total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. The chosen phase 2 dose of PLX3397 was 1000 mg per day. In the extension study, 12 patients with tenosynovial giant-cell tumors had a partial response and 7 patients had stable disease. Responses usually occurred within the first 4 months of treatment, and the median duration of response exceeded 8 months. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment. CONCLUSIONS Treatment of tenosynovial giant-cell tumors with PLX3397 resulted in a prolonged regression in tumor volume in most patients. (Funded by Plexxikon; ClinicalTrials.gov number, NCT01004861.).

Tyrphostins IV—Highly potent inhibitors of EGF receptor kinase. Structure-activity relationship study of 4-anilidoquinazolines

Potent 4-anilido-substituted quinazolines which potently inhibit epidermal growth factor receptor (EGFR) kinase were prepared. Structure-activity relationship studies reveal high sensitivity to substitution at the aniline ring.

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.

Abstract 552: Targeting brain tumors with PLX3397, an inhibitor of the CSF-1 receptor kinase

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cancer growth in the brain incites a neuroinflammatory response that becomes a defining feature of the brain tumor microenvironment. Microglia and macrophages provide important functions that support the invasiveness of glioblastoma. Other cancer types that become metastatic to brain also rely on the microenvironment to support angiogenesis. PLX3397 is a potent inhibitor of the transmembrane tyrosine kinase receptor for colony stimulating factor-1 (CSF-1R). The CSF-1R is required for the differentiation and activation of microglia and macrophages. Oral administration of PLX3397 to mice at 20 mg/kg qd significantly reduces the microglia/macrophage marker, Iba1, as determined by western blotting. PLX3397 penetrates the blood-brain barrier, as determined through pharmacokinetic analysis, with brain levels reaching substantial fractions of the concurrent plasma levels. PLX3397 is highly bound to plasma albumin, and therefore the levels attained in the brain likely affect Iba1 levels through a local inhibition of brain microglia and macrophages, although a peripheral effect may also contribute. Culture of glioblastoma cell lines including U87, and treatment with chemotherapeutic agents or radiation, was found to cause a 4-fold elevation of the two CSF-1R ligands, CSF-1 and IL-34, as quantified by QPCR. This indicates that glioma cells can recruit and stimulate microglia and macrophages, and that current standard therapies likely exacerbate this stimulation. Other cancer types that are known to form metastases to brain, including melanoma and breast cancer, show similar abilities to produce these cytokines in response to standard therapies. The rat 9L glioblastoma line forms an aggressive tumor when tested as an orthotopic model in syngeneic Fisher rats. Seven days after implantation, PLX3397 was administered via rodent chow for 14 days, reducing the tumor growth by 44% as determined by MRI. These results provide preclinical evidence that PLX3397 may show a clinical benefit in brain cancers, either as a single agent or in combination with standard chemo- or radiation therapies. PLX3397 is nearing completion of a successful Phase 1 dose-escalation safety trial in solid tumor cancer patients. 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 552. doi:10.1158/1538-7445.AM2011-552

Abstract 3850: Efficacy of the selective CSF-1R kinase inhibitor PLX3397 in mouse models of tumor growth and bone metastasis

Macrophages of the tumor microenvironment can facilitate cancer progression and are a possible target for therapy. We have developed PLX3397, an orally-active, selective small molecule inhibitor of the colony stimulating factor-1 receptor (CSF-1R) kinase. CSF-1R is known to control a key signaling pathway for the recruitment of macrophages to tumors, and also for differentiation of osteoclasts mediating the destructive processes of bone metastasis. We show efficacy of PLX3397 in three different animal cancer models. PLX3397, which is currently in Phase I clinical trial, also inhibits the related kinase Kit, although we believe the efficacy observed here primarily reflects CSF-1R inhibition. SK-N-SH human neuroblastoma cells grown as xenografts in female nude mice were inhibited 60% in their growth compared to controls after 18 days oral dosing of PLX3397. This contrasts with a very weak effect on growth of SK-N-SH cells in culture, with an IC50 of 10 μM. A similar lack of growth inhibitory effects of PLX3397 was obtained using several cancer cells and when tested in cell toxicity assays. In a separate study, PLX3397 had little or no effect on the growth of MDA-MB-231 human tumor cells grown as xenografts. These results suggest that the observed inhibition of SK-N-SH cells grown in vivo are mediated by targeting cells of the microenvironment, and further, that this neuroblastoma may be more dependant on the microenvironment than other tumor cells. The MMTV-PyMT transgenic mouse breast cancer model has been used previously to identify mechanisms through which tumor-associated macrophages can enhance metastasis. Using this model, PLX3397 caused a 90% reduction in circulating tumor cells (CTCs), quantified by FACS analysis with a pan-cytokeratin antibody. This decrease was observable after two oral doses given at 18 and 4 hours before a terminal blood sample was taken. A similar decrease in CTCs was observed using QPCR in syngeneic mice orthotopically implanted with a tumor cell line derived from the transgenic mice. PLX3397 also worked at low concentrations in vitro to reduce by 55% the ability of macrophages (purified from tumors) to induce invasive acini in breast cancer cells grown in culture as spheroids. MDA-MB-468 human breast tumor cells injected into the tibia of female SCID mice caused trabecular bone loss as measured by micro-CT, and this bone lysis was associated with a 4-fold elevation in osteoclast number. Oral dosing of PLX3397 prevented both the rise in osteoclasts and the loss of bone, suggesting this compound is able to inhibit the intended CSF-1R target in vivo, and predicting it may offer benefit to cancer patients suffering with bone metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3850.