Ensar Halilovic

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.

4E-BP1 Is a Key Effector of the Oncogenic Activation of the AKT and ERK Signaling Pathways that Integrates Their Function in Tumors

PIK3CA and PTEN alterations are common in human cancer, but only a fraction of such tumors are dependent upon AKT signaling. AKT independence is associated with redundant activation of cap-dependent translation mediated by convergent regulation of the translational repressor 4E-BP1 by the AKT and ERK pathways. This provides mechanistic bases for the limited activity of AKT and MEK inhibitors in tumors with comutation of both pathways and the profound synergy observed with combined inhibition. Whereas such tumors are sensitive to a dominant active 4E-BP1 mutant, knockdown of 4E-BP1 expression reduces their dependence on AKT/ERK signaling for translation or survival. Thus, 4E-BP1 plays a prominent role in mediating the effects of these pathways in tumors in which they are activated by mutation.

Relief of Profound Feedback Inhibition of Mitogenic Signaling by RAF Inhibitors Attenuates Their Activity in BRAFV600E Melanomas

BRAF(V600E) drives tumors by dysregulating ERK signaling. In these tumors, we show that high levels of ERK-dependent negative feedback potently suppress ligand-dependent mitogenic signaling and Ras function. BRAF(V600E) activation is Ras independent and it signals as a RAF-inhibitor-sensitive monomer. RAF inhibitors potently inhibit RAF monomers and ERK signaling, causing relief of ERK-dependent feedback, reactivation of ligand-dependent signal transduction, increased Ras-GTP, and generation of RAF-inhibitor-resistant RAF dimers. This results in a rebound in ERK activity and culminates in a new steady state, wherein ERK signaling is elevated compared to its initial nadir after RAF inhibition. In this state, ERK signaling is RAF inhibitor resistant, and MEK inhibitor sensitive, and combined inhibition results in enhancement of ERK pathway inhibition and antitumor activity.

Genomic and Biological Characterization of Exon 4 KRAS Mutations in Human Cancer

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.

PIK3CA mutation uncouples tumor growth and Cyclin D1 regulation from MEK/ERK and mutant KRAS signaling

Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small-molecule MEK inhibitor, decreases MEK/ERK pathway signaling and destabilizes cyclin D1, resulting in significant anticancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor-resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores cyclin D1 expression and G(1)-S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and phosphoinositide 3-kinase mutations are unlikely to respond to the inhibition of the MEK pathway alone but will require effective inhibition of both MEK and phosphoinositide 3-kinase/AKT pathway signaling.

Therapeutic strategies for inhibiting oncogenic BRAF signaling.

Mitogen-activated protein kinase (MAPK) activation is a common property of human cancers and is often due to activating mutations in the BRAF and RAS genes. BRAF kinase domain mutations, the vast majority of which are V600E, occur in approximately 8% of human tumors. These mutations are non-overlapping in distribution with RAS mutations and are observed most frequently in melanoma but also in tumors arising in the colon, thyroid, lung and other sites. V600E BRAF mutation stimulates extracellular signal-regulated kinase (ERK) signaling, induces proliferation and is capable of promoting transformation. Given the frequent occurrence of BRAF mutations in human cancer and the continued requirement for BRAF activity in tumors in which it is mutated, efforts are underway to develop targeted inhibitors of BRAF and its downstream effectors. These agents offer the possibility of greater therapeutic efficacy than the currently available systemic therapies for tumors driven by activating mutations in the MAPK pathway.

The Public Repository of Xenografts Enables Discovery and Randomized Phase II-like Trials in Mice

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.

A distinct p53 target gene set predicts for response to the selective p53–HDM2 inhibitor NVP-CGM097

Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53–HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53–HDM2 inhibitors, such as NVP-CGM097. DOI: http://dx.doi.org/10.7554/eLife.06498.001

Erratum: The Public Repository of Xenografts Enables Discovery and Randomized Phase II-like Trials in Mice (Cancer Cell (2016) 29 (574–586))

Elizabeth C. Townsend, Mark A. Murakami, Alexandra Christodoulou, Amanda L. Christie, Johannes Köster, Tiffany A. DeSouza, Elizabeth A. Morgan, Scott P. Kallgren, Huiyun Liu, Shuo-Chieh Wu, Olivia Plana, Joan Montero, Kristen E. Stevenson, Prakash Rao, Raga Vadhi, Michael Andreeff, Philippe Armand, Karen K. Ballen, Patrizia Barzaghi-Rinaudo, Sarah Cahill, Rachael A. Clark, Vesselina G. Cooke, Matthew S. Davids, Daniel J. DeAngelo, David M. Dorfman, Hilary Eaton, Benjamin L. Ebert, Julia Etchin, Brant Firestone, David C. Fisher, Arnold S. Freedman, Ilene A. Galinsky, Hui Gao, Jacqueline S. Garcia, Francine Garnache-Ottou, Timothy A. Graubert, Alejandro Gutierrez, Ensar Halilovic, Marian H. Harris, Zachary T. Herbert, Steven M. Horwitz, Giorgio Inghirami, Andrew M. Intlekofer, Moriko Ito, Shai Izraeli, Eric D. Jacobsen, Caron A. Jacobson, Sébastien Jeay, Irmela Jeremias, Michelle A. Kelliher, Raphael Koch, Marina Konopleva, Nadja Kopp, Steven M. Kornblau, Andrew L. Kung, Thomas S. Kupper, Nicole R. LeBoeuf, Ann S. LaCasce, Emma Lees, Loretta S. Li, A. Thomas Look, Masato Murakami, Markus Muschen, Donna Neuberg, Samuel Y. Ng, Oreofe O. Odejide, Stuart H. Orkin, Rachel R. Paquette, Andrew E. Place, Justine E. Roderick, Jeremy A. Ryan, Stephen E. Sallan, Brent Shoji, Lewis B. Silverman, Robert J. Soiffer, David P. Steensma, Kimberly Stegmaier, Richard M. Stone, Jerome Tamburini, Aaron R. Thorner, Paul van Hummelen, Martha Wadleigh, Marion Wiesmann, Andrew P. Weng, Jens U. Wuerthner, David A. Williams, Bruce M. Wollison, Andrew A. Lane, Anthony Letai, Monica M. Bertagnolli, Jerome Ritz, Myles Brown, Henry Long, Jon C. Aster, Margaret A. Shipp, James D. Griffin, and David M. Weinstock* *Correspondence: dweinstock@partners.org http://dx.doi.org/10.1016/j.ccell.2016.06.008

Inhibition of Wild-Type p53-Expressing AML by the Novel Small Molecule HDM2 Inhibitor CGM097

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient–derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML. Mol Cancer Ther; 14(10); 2249–59. ©2015 AACR.