Alfonso Zambon

RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.

BACKGROUND Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma

UNLABELLED We generated cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)-SRC family kinase (SFK)-STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo, and monotherapy with the broad specificity tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo. We analyzed tumors from patients with intrinsic or acquired resistance to vemurafenib and observed increased EGFR and SFK activity. Furthermore, dasatinib blocked the growth and metastasis of one of the resistant tumors in immunocompromised mice. Our data show that BRAF inhibitor-mediated activation of EGFR-SFK-STAT3 signaling can mediate resistance in patients with BRAF-mutant melanoma. We describe 2 treatments that seem to overcome this resistance and could deliver therapeutic efficacy in patients with drug-resistant BRAF-mutant melanoma. SIGNIFICANCE Therapies that target the driver oncogenes in cancer can achieve remarkable responses if patients are stratified for treatment. However, as with conventional therapies, patients often develop acquired resistance to targeted therapies, and a proportion of patients are intrinsically resistant and fail to respond despite the presence of an appropriate driver oncogene mutation. We found that the EGFR/SFK pathway mediated resistance to vemurafenib in BRAF -mutant melanoma and that BRAF and EGFR or SFK inhibition blocked proliferation and invasion of these resistant tumors, providing potentially effective therapeutic options for these patients.

Gatekeeper Mutations Mediate Resistance to BRAF-Targeted Therapies

Gatekeeper mutations in BRAF confer drug resistance. Getting Back on Target The identification of clinically useful small-molecule inhibitors of dysregulated proteins that trigger a cascade of detrimental downstream effects during the process of tumorigenesis requires a highly precise understanding of the specific residues responsible for the damage. However, although this information can elucidate promising candidate targets, clinical data have revealed that the utility of anticancer drugs within tumors of similar origin and mutational status is variable. It has been postulated that the haphazard effects of such drugs are the result of off-targeting effects—that is, the ability of a drug to inhibit tumor growth in a manner less specific than that which it was optimally designed for. In an era that now seeks to further personalize medicine, these off-targeting effects reveal how distant we remain from truly achieving an individualized cancer cocktail. In melanoma, the failure of the FDA-approved drug sorafenib to consistently curb disease in individuals with tumors that harbor cancer-driving mutations in BRAF—the overactive kinase presumed to be the target of the drug on the basis of its efficacy against CRAF—raised doubts as to the utility of BRAF as true melanoma target, despite its more successful application as a liver and kidney cancer therapeutic. Now, Marais and colleagues have designed a system by which to tease apart whether BRAF-mediated therapeutics such as sorafenib and PLX4720 inhibit tumor growth by homing in on the gatekeeper mutations that cause aberrant signaling in BRAF or by other nonspecific tumor-inhibiting mechanisms. Their compelling results reveal that BRAF is indeed a tractable target for melanoma, and while a plethora of tractable cancer targets are continuously being resolved, the need for proper preclinical validation—where the effects can easily be discerned as being mechanistic or off-target—is paramount to developing the right drug for the specific target. BRAF is a serine-threonine–specific protein kinase that is mutated in 2% of human cancers. Oncogenic BRAF is a validated therapeutic target that constitutively activates mitogen-activated protein kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) signaling, driving tumor cell proliferation and survival. Drugs designed to target BRAF have been developed, but it is difficult to prove that they mediate their antitumor effects by inhibiting BRAF rather than by working through off-target effects. We generated drug-resistant versions of oncogenic BRAF by mutating the gatekeeper residue. Signaling by the mutant proteins was resistant to the small-molecule inhibitor sorafenib, but sorafenib still inhibited the growth of tumors driven by the mutant protein. In contrast, both BRAF signaling and tumor growth were resistant to another RAF drug, PLX4720. These data provide unequivocal evidence that sorafenib mediates its antitumor effects in a manner that is independent of its ability to target oncogenic BRAF, whereas PLX4720 inhibits tumor growth by targeting oncogenic BRAF directly.

Paradox-Breaking RAF Inhibitors that Also Target SRC Are Effective in Drug-Resistant BRAF Mutant Melanoma

Summary BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.

Development and exploitation of CK2 inhibitors

A number of quite specific and fairly potent inhibitors of protein kinase CK2, belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines are available to date. The structural basis for their selectivity is provided by a hydrophobic pocket adjacent to the ATP/GTP binding site, which in CK2 is smaller than in the majority of other protein kinases due to the presence of a number of residues whose bulky side chains are generally replaced by smaller ones. Consequently a doubly substituted CK2 mutant V66A,I174A is much less sensitive than CK2 wild type to these classes of inhibitors. The most efficient inhibitors both in terms of potency and selectivity are 4,5,6,7-tetrabromo-1H-benzotriazole, TBB (Ki = 0.4 μM), the TBB derivative 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, DMAT (Ki = 0.040 μM), the emodin related coumarinic compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2-one, NBC (Ki = 0.22 μM) and the indoloquinazoline derivative ([5-oxo-5,6-dihydroindolo-(1,2a)quinazolin-7-yl]acetic acid), IQA (Ki = 0.17 μM). These inhibitors are cell permeable as judged from ability to block CK2 in living cells and they have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signaling pathways affected by CK2 and to identify the endogenous substrates of this pleitropic kinase. By blocking CK2 these inhibitors display a remarkable pro-apoptotic efficacy on a number of tumor derived cell lines, a property which can be exploited in perspective to develop antineoplastic drugs.

BRAF Inhibitors Induce Metastasis in RAS Mutant or Inhibitor-Resistant Melanoma Cells By Reactivating MEK and ERK Signaling

When therapy leads to cancer metastasis, knowing where else to target in the pathway may be the key to successful treatment. Blocking Melanoma Metastasis Although inhibitors of the mutant BRAF kinase are effective in some melanoma patients, intrinsic or acquired resistance to the drug is common. Furthermore, the growth of melanoma tumors with concomitant mutations in guanosine triphosphatase RAS, which activated kinases in the RAF family, is paradoxically accelerated by BRAF inhibition. RAF is the first kinase in a three-kinase cascade [the RAF–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal–regulated kinase) pathway] that is involved in cell proliferation. Using proteomics, patient material, and mouse models, Sanchez-Laorden et al. found that BRAF inhibition paradoxically stimulated MEK and ERK signaling to induce metastasis of melanoma cells with mutant BRAF, resistance to a BRAF inhibitor, or mutant RAS. Combined treatment with a MEK inhibitor prevented BRAF inhibitor–induced metastasis in mice. Thus, combination therapies may be best to prevent both primary tumor growth and drug-induced metastasis. Melanoma is a highly metastatic and lethal form of skin cancer. The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF–ERK (extracellular signal–regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)–ERK pathway, increased expression and secretion of interleukin 8, and induction of protease-dependent invasion. These events were accompanied by a cell morphology switch from predominantly rounded to predominantly elongated cells. We also observed similar responses in BRAF inhibitor–resistant melanoma cells. These data show that BRAF inhibitors can induce melanoma cell invasion and metastasis in tumors that develop resistance to these drugs.

Small molecule inhibitors of BRAF in clinical trials

Over the last few years, BRAF has emerged as a validated target in melanoma. This review summarises recent advances in the development of BRAF inhibitors, focussing on agents that have been assessed clinically.

Synthesis, Structure-Activity Relationship and Crystallographic Studies of 3-Substituted Indolin-2-One Ret Inhibitors.

The synthesis, structure-activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers.

Surface acidity modifications induced by thermal treatments and acid leaching on microcrystalline H-BEA zeolite. A FTIR, XRD and MAS-NMR study

Samples of beta-zeolite thermally treated at different temperatures and acid leached with diluted hydrochloric acid solution have been investigated by XRD, 27Al MAS-NMR spectroscopy and FTIR spectroscopy. The results have been used to interpret the behaviour of the samples as catalysts for the acylation of 2-methoxy-naphthalene. It has been shown that the as prepared sample presents two types of extraframework species. These are identified as Al hydroxo-ions highly dispersed in the internal zeolite channels and Al oxide nanoparticles. Calcination causes dealumination of the framework and progressive conversion of the Al hydroxo-ions into Al oxide nanoparticles that reduce the zeolite channels practicability, modifying the shape selectivity effect. The Bronsted acid sites present in the sinusoidal channels (νOH=3608 cm−1) can be distinguished from those located in the larger ones (νOH=3620–3612 cm−1), due to the inability of the bulky probe molecule pivalonitrile to enter the former. However, internal terminal silanols (νOH=3735 cm−1) also apparently display a significant Bronsted acidity, definitely higher than the acidity of those absorbing at 3747 cm−1, thought to be located at the external crystal surface. The strongest Lewis acidity is displayed by the aluminum hydroxo-ions, while that of alumina nanoparticles is a little weaker. Acid-leached beta-zeolite also displays a medium strength Lewis acidity, likely due to framework Al cations. Thus a partial reinterpretation of the real structure of beta-zeolite and a partial reassignment of the bands due to the surface hydroxy groups are proposed.

Novel potent BRAF inhibitors: toward 1 nM compounds through optimization of the central phenyl ring.

BRAF, a serine/threonine specific protein kinase that is part of the MAPK pathway and acts as a downstream effector of RAS, is a potential therapeutic target in melanoma. We have developed a series of small-molecule BRAF inhibitors based on a 1H-imidazo[4,5-b]pyridine-2(3H)-one scaffold (ring A) as the hinge binding moiety and a number of substituted phenyl rings C that interact with the allosteric binding site. The introduction of various groups on the central phenyl ring B combined with appropriate A- and C-ring modifications afford very potent compounds that inhibit (V600E)BRAF kinase activity in vitro and oncogenic BRAF signaling in melanoma cells. Substitution on the central phenyl ring of a 3-fluoro, a naphthyl, or a 3-thiomethyl group improves activity to yield compounds with an IC(50) of 1 nM for purified (V600E)BRAF and nanomolar activity in cells.