James H. Tsai

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.

The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner

Tumors with mutant BRAF and some with mutant RAS are dependent upon ERK signaling for proliferation, and their growth is suppressed by MAPK/ERK kinase (MEK) inhibitors. In contrast, tumor cells with human EGF receptor (HER) kinase activation proliferate in a MEK-independent manner. These findings have led to the development of RAF and MEK inhibitors as anticancer agents. Like MEK inhibitors, the RAF inhibitor PLX4032 inhibits the proliferation of BRAFV600E tumor cells but not that of HER kinase-dependent tumors. However, tumors with RAS mutation that are sensitive to MEK inhibition are insensitive to PLX4032. MEK inhibitors inhibit ERK phosphorylation in all normal and tumor cells, whereas PLX4032 inhibits ERK signaling only in tumor cells expressing BRAFV600E. In contrast, the drug activates MEK and ERK phosphorylation in cells with wild-type BRAF. In BRAFV600E tumor cells, MEK and RAF inhibitors affect the expression of a common set of genes. PLX4032 inhibits ERK signaling output in mutant BRAF cells, whereas it transiently activates the expression of these genes in tumor cells with wild-type RAF. Thus, PLX4032 inhibits ERK signaling output in a mutant BRAF-selective manner. These data explain why the drug selectively inhibits the growth of mutant BRAF tumors and suggest that it will not cause toxicity resulting from the inhibition of ERK signaling in normal cells. This selectivity may lead to a broader therapeutic index and help explain the greater antitumor activity observed with this drug than with MEK inhibitors.

Biology of Diaphorina citri (Homoptera: Psyllidae) on Four Host Plants

Abstract The biology of the citrus psyllid Diaphorina citriKuwayama was studied at 25°C on four commonly grown citrus andrelated plants [rough lemon, Citrus jambhiri Lush; sourorange, C. aurantium L.; grapefruit, C. paradisiMacfadyen; and orange jessamine, Murraya paniculata (L.)Jack] in the laboratory. The biological characteristics of each lifestage are described. The average egg incubation periods on orangejessamine, grapefruit, rough lemon, and sour orange varied very little(4.1–4.2 d). The average nymphal developmental periods on these fourhost plants were essentially the same except the fifth stadium.Survival of immatures on orange jessamine, grapefruit, rough lemon, andsour orange was 75.4, 84.6, 78.3, and 68.6%, respectively. Femaleadults lived an average of 39.7, 39.7, 47.6, and 43.7 d on theserespective host plants. The average number of eggs laid per female ongrapefruit (858 eggs) was significantly more than those on other hosts(P < 0.05). The intrinsic rate of natural increase(rm) for D. citri on grapefruit washighest. Jackknife estimates of rm varied from0.188 on grapefruit to 0.162 on orange jessamine and rough lemon. Themean population generation time on these hosts ranged from 31.6 to34.1 d. The continuous flushes produced by orange jessamine couldplay an important role in maintaining high populations of this vectorwhen the new flushes are not available in the commercial citrus groves.

PLX4032, a Potent Inhibitor of the B-Raf V600E Oncogene, Selectively Inhibits V600E-positive Melanomas

Targeted intervention of the B‐Raf V600E gene product that is prominent in melanoma has been met with modest success. Here, we characterize the pharmacological properties of PLX4032, a next‐generation inhibitor with exquisite specificity against the V600E oncogene and striking anti‐melanoma activity. PLX4032 induces potent cell cycle arrest, inhibits proliferation, and initiates apoptosis exclusively in V600E‐positive cells in a variety of in vitro experimental systems; follow‐up xenograft studies demonstrate extreme selectivity and efficacy against melanoma tumors bearing the V600E oncoproduct. The collective data support further exploration of PLX4032 as a candidate drug for patients with metastatic melanoma; accordingly, validation of PLX4032 as a therapeutic tool for patients with melanoma is now underway in advanced human (Phase III) clinical trials.

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.

SEASONAL ABUNDANCE OF THE ASIAN CITRUS PSYLLID, DIAPHORINA CITRI (HOMOPTERA: PSYLLIDAE) IN SOUTHERN FLORIDA

Abstract Seasonal abundance of the Asian citrus psyllid, Diaphorina citri Kuwayama, was studied weekly in two orange jasmine [Murraya paniculata (L.) Jack] plots in southern Florida from October 1998 to October 1999. Psyllid populations occur throughout the season on orange jasmine in southern Florida. Population peaks were observed in October, November, and December in 1998, and May and August in 1999. Psyllid population levels were positively related to the availability of new shoot flushes which were in turn related to the weekly minimum temperature and rainfall. Natural enemies were not key factors in regulating populations during the study period. The populations of adult psyllids were also studied weekly on potted orange jasmine and grapefruit (Citrus paradisi Macfadyen) plants from June 1999 to July 2000. The population levels of psyllid on both host plants were not significantly different and general population trends on the two hosts were similar over time. Continuous shoot flushes produced by orange jasmine could play an important role in maintaining high populations of this insect when new shoot flushes were not available in the commercial citrus groves.

Effects of Host Plants on Biology and Life Table Parameters of Aphis spiraecola (Homoptera: Aphididae)

Abstract The development, survivorship, longevity, reproduction, and life table parameters of the spirea aphid, Aphis spiraecola Patch, were evaluated at 25°C on seven commonly grown plants (Polyscias crispata (Bull) Merrill cultivar chicken-gizzard aralia, P. scutellaria (Burman) Fosberg, Vibernum suspensum Lindley, grapefruit (Citrus paradisi Macfadyen), rough lemon (C. jambhiri Lushington), pineapple orange [C. sinensis (L.) Osbeck variety pineapple orange], orange jessamine [Murraya paniculata (L.) Jack]) in the laboratory. Spirea aphid failed to survive on orange jessamine. The developmental times for the immature stages ranged from 7.9 d on P. scutellaria to 9.9 d on grapefruit or pineapple orange. The immature survival varied from 92.7% on P. scutellaria to 78.8% on pineapple orange. The average number of nymphs reproduced per female were 42.7, 35.7, 28.7, 22.3, 18.0, and 11.7 on P. crispata, P. scutellaria, V. suspensum, grapefruit, rough lemon, and pineapple orange, respectively. Female adults lived an average of 14.1, 15.3, 12.2, 11.0, 12.0, and 9.3 d on these same hosts. The intrinsic rate of increase (rm) for spirea aphid on P. crispata was highest. Jackknife estimates of rm varied from 0.308 on P. crispata to 0.177 on pineapple orange. The mean population generation time on these hosts ranged from 11.6 to 13.2 d. It was concluded that the ability of spirea aphid to feed and develop on a wide range of host plants increases its chance to infest citrus and thereby spreading the citrus tristeza virus.

Sampling of Diaphorina citri (Homoptera: Psyllidae) on orange jessamine in southern Florida.

Dispersion indices and related statistics of Asian citrus psyllid, Diaphorina citri Kuwayama, on orange jessamine [Murraya paniculata (L.) Jack] shoots in southern Florida from 1998 to 1999 were determined with 235 data sets and used to develop sampling plans. Three regression models, Taylors power law, Iwaos patchiness regression, and k = c +dm [k = m2 / (S2 - m)] (where k is the parameter for the negative binomial distribution) were used to analyze the data. Taylors power law (a = 0.3407 0.03, b = 1.2971 0.03, r2 = 0.88) fit the data better than Iwaos model (a = -0.3217 0.12, = 1.6979 0.06, r2 = 0.76). Taylors b and Iwaos were both significantly > 1, indicating that D. citri populations were aggregated. Iwaos a was significantly