BRAF Activation Research Articles

Abstract A63: IFNAR1 downregulation during melanoma progression upregulates αv expression and promotes metastasis

Abstract The anti-metastatic effects of type 1 interferon (IFN1) prompted its use for adjuvant therapy in malignant melanoma patients at high risk for developing metastasis. While IFN1 adjuvant therapy continues to be the preferred treatment, many patients fail to elicit response to IFN1 treatment and progress to develop distal metastases often resulting in a lethal outcome. We observed downregulation of the IFNAR1 chain of the IFN1 receptor and suppression of IFN1 signaling in genetically engineered mouse melanomas driven by melanocyte-specific activation of Braf and inactivation of Pten. Induction of melanoma in animals expressing IFNAR1 that is deficient in ubiquitination and degradation led to a delayed onset of the disease and decreased number of local metastases. Remarkably, no distant metastases were found in these mice despite substantial primary tumor burden. Gene set enrichment analysis demonstrated a dramatic loss of the αv integrin pathway signature in mice harboring stabilized IFNAR1. Conversely, melanomas induced in Ifnar1 knockout mice yielded transplantable cell lines that displayed high levels of αv expression. Restoring IFNAR1 expression in these cells led to the loss of αv expression. We discuss the role of these mechanisms in melanoma metastasis and refractoriness to IFN1-based therapy. Citation Format: Angelica Ortiz, Yuliya V. Katlinskaya, Kanstantsin V. Katlinski, Serge Y. Fuchs. IFNAR1 downregulation during melanoma progression upregulates αv expression and promotes metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A63.

The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells.

In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.

BRAF and MAP2K1 mutations in hairy cell leukemia and splenic marginal zone B-cell lymphoma

DNA samples from patients with hairy cell leukemia (HCL) and splenic marginal zone B-cell lymphoma (MZBCL) for BRAF and MAP2K1 activating mutations were analyzed. V600E mutation of BRAF was detected in 39 (98 %) of 40 patients with hairy cell leukemia, and no patient with MZBCL. In none of the patients in this study any other activating mutations than V600E in exons 11 and 15 of BRAF gene were revealed. MAP2K1 Q56P mutation characterized by IGHV4-34 expression was detected in 1 (2 %) of 40 patients with HCL. In none of the patients with MZBCL activating mutations in the 2, 3 or 11 exons of MAR2K1 gene have been identified, including those with IGHV4-34 expression.

Optogenetically controlled RAF to characterize BRAF and CRAF protein kinase inhibitors.

Here, we applied optoRAF, an optogenetic tool for light-controlled clustering and activation of RAF proteins that mimics the natural occurring RAS-mediated dimerization. This versatile tool allows studying the effect on BRAF and CRAF homodimer- as well as heterodimer-induced RAF signaling. Vemurafenib and dabrafenib are two clinically approved inhibitors for BRAF that efficiently suppress the kinase activity of oncogenic BRAF (V600E). However in wild-type BRAF expressing cells, BRAF inhibitors can exert paradoxical activation of wild-type CRAF. Using optoRAF, vemurafenib was identified as paradoxical activator of BRAF and CRAF homo- and heterodimers. Dabrafenib enhanced activity of light-stimulated CRAF at low dose and inhibited CRAF signaling at high dose. Moreover, dabrafenib increased the protein level of CRAF proteins but not of BRAF proteins. Increased CRAF levels correlate with elevated RAF signaling in a dabrafenib-dependent manner, independent of light activation.

Inhibition of oncogenic BRAF activity by indole-3-carbinol disrupts microphthalmia-associated transcription factor expression and arrests melanoma cell proliferation.

Indole-3-carbinol (I3C), an anti-cancer phytochemical derived from cruciferous vegetables, strongly inhibited proliferation and down-regulated protein levels of the melanocyte master regulator micropthalmia-associated transcription factor (MITF-M) in oncogenic BRAF-V600E expressing melanoma cells in culture as well as in vivo in tumor xenografted athymic nude mice. In contrast, wild type BRAF-expressing melanoma cells remained relatively insensitive to I3C anti-proliferative signaling. In BRAF-V600E-expressing melanoma cells, I3C treatment inhibited phosphorylation of MEK and ERK/MAPK, the down stream effectors of BRAF. The I3C anti-proliferative arrest was concomitant with the down-regulation of MITF-M transcripts and promoter activity, loss of endogenous BRN-2 binding to the MITF-M promoter, and was strongly attenuated by expression of exogenous MITF-M. Importantly, in vitro kinase assays using immunoprecipitated BRAF-V600E and wild type BRAF demonstrated that I3C selectively inhibited the enzymatic activity of the oncogenic BRAF-V600E but not of the wild type protein. In silico modeling predicted an I3C interaction site in the BRAF-V600E protomer distinct from where the clinically used BRAF-V600E inhibitor Vemurafenib binds to BRAF-V600E. Consistent with this prediction, combinations of I3C and Vemurafenib more potently inhibited melanoma cell proliferation and reduced MITF-M levels in BRAF-V600E expressing melanoma cells compared to the effects of each compound alone. Thus, our results demonstrate that oncogenic BRAF-V600E is a new cellular target of I3C that implicate this indolecarbinol compound as a potential candidate for novel single or combination therapies for melanoma. © 2016 Wiley Periodicals, Inc.

Discovery of EBI-907: A highly potent and orally active B-Raf(V600E) inhibitor for the treatment of melanoma and associated cancers.

A novel series of pyrazolo[3,4-c]isoquinoline derivatives was discovered as B-Raf(V600E) inhibitors through scaffold hopping based on a literature lead PLX4720. Further SAR exploration and optimization led to the discovery of potent B-Raf(V600E) inhibitors with good oral bioavailability in rats and dogs. One of the compounds EBI-907 (13g) demonstrated excellent in vivo efficacy in B-Raf(V600E) dependent Colo-205 tumor xenograft models in mouse and is under preclinical studies for the treatment of melanoma and B-Raf(V600E) associated cancers.

Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants.

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf(AVKA) mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf(AVKA) allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf(AVKA) mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf(AVKA). Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.

Cooperative Activity of BRAF F595L and Mutant HRAS in Histiocytic Sarcoma Provides New Insights into Oncogenic BRAF Signaling

Activating BRAF mutations, in particular V600E/K, drive many cancers, including a substantial proportion of systemic histiocytic disorders, and mutant BRAF-selective inhibitors are promising therapeutics for these diseases. Activating BRAF alleles are considered mutually exclusive with mutations in RAS family members, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif can coexist with oncogenic RAS and cooperate via transactivation of wildtype RAF proteins and paradoxical MEK/ERK activation. Due to the increasing use of global approaches to tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We used several in vitro experimental systems, including Braf-deficient murine embryonic fibroblasts expressing a regulatable HRAS oncogene, to determine the biochemical properties and cellular effects of a largely uncharacterized mutation, F595L, in the DFG motif of the BRAF activation segment that was identified by clinical exome sequencing in a patient with histiocytic sarcoma and multiorgan involvement and also occurs as somatic alteration in colorectal adenoma or carcinoma, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, melanoma, and neuroblastoma and as germline mutation in cardio-facio-cutaneous syndrome. In addition, we investigated the interaction between BRAF F595L and a concomitant HRAS Q61R allele, which was present in the same tumor cell clone and occurs as acquired alteration in multiple tumor types and as inherited variant in Costello syndrome. Unlike previously described DFG motif mutants, BRAF F595L is a gain-of-function variant with intermediate activity towards MEK that, in sharp contrast to BRAF V600E, requires an intact dimer interface for downstream signaling. Furthermore, BRAF F595L does not act paradoxically, but nevertheless cooperates with mutant HRAS to induce maximal activity of the MEK-ERK signaling pathway. Of immediate clinical relevance, BRAF F595L shows divergent responses to the mutant BRAF-selective inhibitors vemurafenib and dabrafenib, whereas signaling driven by BRAF F595L with and without mutant HRAS is efficiently blocked by the pan-RAF inhibitors sorafenib and AZ628 and the MEK inhibitor trametinib. Consistent with this, sorafenib treatment led to abrogation of aberrant MEK/ERK signaling in the index case with histiocytic sarcoma driven by BRAF F595L and HRAS Q61R. Mutation data from patients and cell lines, representing 18 different tumor entities, show that BRAF F595L as well as other BRAF mutants with intermediate signaling activity coincide with mutant RAS in at least 40% and 23% of cases, respectively. These data define a distinct class of activating BRAF mutations that cooperate with oncogenic RAS in a non-paradoxical fashion, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway, and underscore the value of comprehensive genomic profiling for uncovering the vulnerabilities of individual tumors. DisclosuresOff Label Use: Administration of sorafenib in a patient with histiocytic sarcoma.

Abstract A2-29: A PDK1 inhibitors that has antitumor activity in a vemurafenib resistant BRAF(V600E)::NRAS(G12V) melanoma PDX

Abstract Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling is activated in many cancers, and is associated with mutational loss of PTEN function, or activation of Phosphatidylinositide-3-kinase (PI3K). Activation of the PDPK1 signaling pathway contributes to proliferation and survival pathways in tumor cells. In a mouse Braf(V600E)::Pten(-/-) melanoma model genetic inactivation of PDKP1 delays the development of pigmented lesions and melanoma. We have found that the PDPK1 inhibitor PHT-427 has antitumor activity in BRAF(V600E)::NRAS(G12V) melanoma patient derived xenografts (PDX) grown in immune deficient mice. Pleckstrin homology (PH) domains are 100-120 amino acid domains found in more than 250 human proteins that bind phosphatidylinositide (Ptdlns) lipids in cell membranes. The phosphorylation of Ptdins, and the consequent binding of PH domain containing proteins is found in many signal transduction pathways critical for cell growth, survival, angiogenesis, and metastasis. Though the primary amino acid sequences of PH domains are not highly conserved, PH domains do have a highly conserved tertiary structure. This structural conservation of PH domains combined with the role PH domains play in signal transduction make them promising targets for small molecule inhibitors. PHT-427 was designed to bind PH domains, and experiments show that PHT-427 binds with the highest affinity to the PH domain of PDKP1. We wanted to test the antitumor activity of PHT-427 in human melanoma PDX models of varying genetic background. About 50% of melanoma tumors are driven by the BRAF(V600E) mutation, and these tumors are effectively targeted with BRAF inhibitors, such as vemurafenib. Unfortunately, these tumors usually develop resistance to vemurafenib, and the disease progresses. Additionally, 40-50% of melanoma tumors do not carry the BRAF(V600E) mutation, indicating the need to target additional pathways in melanoma. We sequenced human PDX samples on the Ion Proton sequencer, using the 409 gene Comprehensive Cancer Panel from Life Technologies. Compared to xenografts, PDXs are closer to the tumor biology of the patient, having a higher degree of molecular subtypes and intratumor heterogeneity, and a mixture of human and mouse stroma. The TMAP alignment program that works with Ion Torrent sequence data cannot handle a combined human-mouse reference genome, we developed a set of scripts we call Graft Extraction Out of REcipient Genome (GEORGE). Sequence reads aligned to the hg19 human reference, and mm10 mouse reference are evaluated by GEORGE and assigned to either the human, or mouse genome. In this project, only human assigned reads were used for variant calling. We sequence 14 melanoma samples, and five samples carried BRAF(V600E). We tested the antitumor activity of PHT-427 in four PDX samples with various BRAF and NRAS mutational statuses. We found that PHT-427 had antitumor activity in a vemurafenib resistant BRAF(V600E)::NRAS(G12V) mutant PDX sample. Citation Format: Brian P. James, Robert Lemos, Jr., Stacey Bagby, Steven Robinson, William Robinson, John Tentler, S. Gail Eckhardt, D. Lynn Kirkpatrick, Garth Powis. A PDK1 inhibitors that has antitumor activity in a vemurafenib resistant BRAF(V600E)::NRAS(G12V) melanoma PDX. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-29.

Effects of a novel Nodal-targeting monoclonal antibody in melanoma.

Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.

Abstract A77: Differential recruitment of myeloid derived immune cells and fibroblasts to the thyroid tumor microenvironment in mouse models of papillary and follicular thyroid cancer

Abstract Thyroid cancer is the most common endocrine malignancy, and its incidence has steadily increased worldwide over the last 30 years. Genetic alterations leading to the activation of the MAPK signaling pathway are crucial for the initiation and progression of thyroid cancer, as evidenced by the high frequency of activating mutations in BRAF, HRAS, NRAS, RET and TRK. Mutations in BRAF are observed in ~45% of papillary thyroid cancers (PTCs), while follicular thyroid cancer (FTC) is often associated with mutations in RAS. We have shown that thyroid specific expression of HrasG12V and Pten inactivation leads to the development of FTCs and poorly differentiated thyroid cancer (PDTC) in HrasG12V/Ptenhom/TPO-cre mice while thyroid specific expression of BrafV600E and loss of Pten leads to the rapid development of PTC and PDTC in BrafV600E/Ptenhom/TPO-cre mice with complete lethality by weaning. We sought to determine whether activation of Braf or Hras resulted in the differential recruitment of stromal cells to the thyroid tumor microenvironment in HrasG12V/Ptenhom/TPO-cre and BrafV600E/Ptenhom/TPO-cre mice. FACS analysis revealed a significant increase in the overall amount of CD45+ cells in Hras (60.95±5.28%) versus Braf (18.35±1.3%) driven murine thyroid tumors. However, we found that the population of CD45+ cells in Braf driven tumors was comprised of a higher percentage of CD11b+ myeloid cells (62.1±1.3%) in comparison to Hras (37.0±5.5%) driven tumors. We next measured levels of cytokine expression in tumor cell lines derived from BrafV600E/Ptenhom/TPO-cre and HrasG12V/Ptenhom/TPO-cre mice. Braf driven tumor cells expressed higher levels of GM-CSF in comparison to Hras driven tumor cells, which we hypothesize contributes to the larger myeloid compartment observed in BrafV600E/Ptenhom/TPO-cre thyroid tumors. Studies are ongoing to further characterize the immune cell populations in these tumors. FACS analysis also revealed an increase in CD45-CD326-CD140α+ fibroblast infiltrate in Braf versus Hras driven thyroid tumors (68.4±0.424% versus 9.8±4.27%, p<.0001). Fibroblasts are a major source of type I collagen (Col1a1), which has been linked to the immunosuppression of tumor infiltrating leukocytes via inhibition of Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). Therefore, we hypothesized that the increase in fibroblast infiltrate observed in Braf driven thyroid tumors would result in a subsequent increase in Col1a1 production, thus potentially leading to a more immunosuppressive thyroid tumor microenvironment in PTC. Indeed, levels of Col1a1 gene expression were significantly upregulated an average of 80 fold (p<.0001) in Braf versus Hras driven thyroid tumors, with no significant difference in Col1a1 expression between Hras driven tumors and WT thyroid controls. Histopathological analysis via Picrosirius red staining confirmed higher levels of total and fibrillar collagen in Braf versus Hras driven tumors. In addition, microarray analysis of human thyroid tumors revealed that Col1a1 expression levels are highest in PTCs, particularly in those with mutations in BRAF, and lowest in FTCs and follicular variant PTC, which are both associated with a high RAS mutation rate. Because FTCs and PTCs have distinct pathological features and differ in prognosis and responses to therapy, we aim to translate these findings to the discovery of more personalized diagnostic and treatment strategies that are targeted toward the thyroid tumor microenvironment. Citation Format: Lee Ann King, Sergey Novitsky, Julio Ricarte-Filho, Philip Owens, Aime T. Franco. Differential recruitment of myeloid derived immune cells and fibroblasts to the thyroid tumor microenvironment in mouse models of papillary and follicular thyroid cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A77.

CDKN2B Loss Promotes Progression from Benign Melanocytic Nevus to Melanoma.

Deletion of the entire CDKN2B-CDKN2A gene cluster is among the most common genetic events in cancer. The tumor-promoting effects are generally attributed to loss of CDKN2A-encoded p16 and p14ARF tumor suppressors. The degree to which the associated CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here, we show that CDKN2B is highly upregulated in benign melanocytic nevi, contributes to maintaining nevus melanocytes in a growth-arrested premalignant state, and is commonly lost in melanoma. Using primary melanocytes isolated directly from freshly excised human nevi naturally expressing the common BRAF(V600E)-activating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAF(V600E), we show that BRAF activation results in reversible, TGFβ-dependent, p15 induction that halts proliferation. Furthermore, we engineer human skin grafts containing nevus-derived melanocytes to establish a new, architecturally faithful, in vivo melanoma model, and demonstrate that p15 loss promotes the transition from benign nevus to melanoma. Although BRAF(V600E) mutations cause melanocytes to initially proliferate into benign moles, mechanisms responsible for their eventual growth arrest are unknown. Using melanocytes from human moles, we show that BRAF activation leads to a CDKN2B induction that is critical for restraining BRAF oncogenic effects, and when lost, contributes to melanoma.

KRAS, BRAF and gastric cancer

Gastric cancer (GC) remains a major worldwide health problem and survival rates continue to be poor in patients with advanced stage disease despite multimodal treatment combining different chemo(radio)therapy regimens with surgery or best supportive care. Thus, there is an urgent clinical need to identify new potential drug targets in order to improve survival for GC patients. KRAS encodes a small guanosine triphosphatase and point mutations in codons 12 and 13 of KRAS have been detected in many human cancers. BRAF is a member of the RAF family of protein kinases and has a hotspot for mutations in codon 600 (so called V600E mutation). KRAS and BRAF proteins are both components of the MAPK/ ERK pathway. When mutated, KRAS becomes constitutively active resulting in enhanced BRAF activity. KRAS and BRAF mutations in colorectal cancers (CRC) are known predictors of poor response to epidermal growth factor receptor (EGFR) targeting agents. This PubMed and Web of Science based review aimed to analyze and summarize the current literature on mutations in KRAS and BRAF in GC and their relationship to clinicopathological and molecular variables including KRAS amplification. In total, 69 studies were included in this review. The median incidence of a KRAS mutation was 6.5% ranging from 0-29%. The median incidence of KRAS mutations was similar in studies from the East and the West (East: 6%, ranging from 0-20%; West 7.5%, ranging from 0-29%). KRAS amplifications were reported at an incidence of 1-9%. The median BRAF mutation incidence in GC was 0%, ranging from 0% to 12%. Due to the low incidence and often small study size, many of the published studies had insufficient statistical power to detect a potential relationship between KRAS mutation status and clinicopathological variables including patient survival. In summary, the current literature on KRAS and BRAF in GC is still limited and very heterogeneous making any comparisons between different studies difficult. BRAF V600E mutations are very rare in GC. Interestingly, the incidence of KRAS mutations in GC is much lower than that in CRC and there appears to be no difference by ethnicity of the patients. KRAS mutations and KRAS amplifications seem to be mutually exclusive suggesting the need to screen GC patients for both genetic aberrations. So far, all clinical studies in unselected patients with metastatic GC have failed to show a significant benefit for EGFR targeting therapy. However, there has been a recent report indicating that the subgroup of signet ring cell GC, which is known to be resistant to standard cytotoxic chemotherapy, has a higher incidence of KRAS mutations (15%). Thus, EGFR targeted therapy in this particular histological subtype of GC could potentially be a promising treatment option in the future.

Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

Cell hierarchies in colorectal cancer: focus on APC and BRAF.

The Wnt-APC-β-Catenin and RAS-RAF-MAPK signaling pathways serve to maintain homeostasis in the normal intestinal epithelium. This state is characterized by an equilibrium between stemness, proliferation, differentiation and cell death [1]. Recent studies by our group [2] and by Dow et al. [3] elucidate how oncogenes and tumor suppressors influence cell hierarchies during colorectal cancer (CRC) formation in the mouse and highlight the potential of this information for future approaches to cancer therapy. A majority of CRCs are initiated by APC mutations promoting Wnt/β-Catenin activity and stem cell fate [4]. As we described already in 2012, reversible transgenic expression of oncogenic β-Catenin can switch mouse intestinal epithelium between normal and adenomatous cell hierarchies [5]. For these experiments, we used organotypic cell cultures maintaining stem, proliferative and differentiated cells. β-Catenin promoted a universal capacity for self-renewal and expression of cancer stem cell markers, similar to cultures derived from APC−/− adenoma. Withdrawal of oncogenic β-Catenin re-established normal cell hierarchies driven by only few stem cells in the crypts. A new publication by Dow et al. [3] shows that reversible knockdown of APC in the intestine of mice establishes adenomatous growth in vivo, while restoration of APC results in reversion to normal cell hierarchies and tumor regression. When the authors combined reversible APC knockdown with conditional gain- and loss-of-function alleles for the KRAS oncogene and the p53 tumor suppressor, respectively, invasive colon carcinoma developed. Significantly, restoration of APC resulted in complete remission and reverted colon cancer tissue to normal intestinal epithelium even in the presence of oncogenic KRAS and absence of p53. These results demonstrate a surprising ability of intestinal epithelium to regain homeostasis. In contrast to Wnt/β-Catenin, the roles of MAPK activity in the control of intestinal cell hierarchies are not well established. EGFR-RAS-RAF-MAPK transduces crucial proliferative cues in the intestinal crypt. Oncogenic KRAS has a tumor-promoting role in conventional adenoma initiated by loss of APC, while oncogenic BRAF can initiate another type of CRC precursor, termed serrated adenoma, independently of APC [4]. We recently found that transgenic expression of oncogenic BRAF caused intestinal serration, but also abrogated maintenance of stem cells in the mouse intestine, which collectively converted to proliferative progenitors [2]. How can BRAF act as a CRC initiator despite its negative effect on stem cell support? The answer comes from a publication dissecting multi-step CRC development after oncogenic BRAF knock-in in the mouse [6]: activation of BRAF resulted in generalized intestinal hyperplasia with a reduced stem cell pool, and only progressive foci displayed activating mutations in the Wnt/β-Catenin pathway, nuclear β-Catenin and increased gene expression of Wnt targets and stem cell markers. This suggests that increased β-Catenin activity can supplement the stem cell pool after oncogenic activation of BRAF. Indeed, we found that activation of β-Catenin partially protected intestinal tissue from the deleterious effects of BRAF overexpression in vitro and in vivo [2]. Taken together, these results give insight into the mechanisms controlling cell hierarchies in CRC, and suggest that the Wnt/β-Catenin and MAPK signaling pathways must be activated in a coordinate manner to allow for CRC initiation and progression. Figure 1 Balancing of the stem cell pool in mouse models of intestinal cancer development How can constraints on cell hierarchies be exploited for novel cancer therapies? Dow et al. argue that cancer remission upon APC restoration provides a strong case for the Wnt signaling pathway as a therapeutic target [4]. Panels of organotypic cultures derived from human CRC have recently become available [7]. Thus, restoration of APC in human canceroids could provide further evidence for the validity of the concept proposed by Dow and colleagues. Therapies targeting the EGFR-MAPK axis are already in clinical use and in clinical trials, such as EGFR-inhibitors and combinatorial inhibition within the extended MAPK network [4]. Future strategies could take into account vulnerabilities of the stem cell pool towards MAPK signals in certain types of CRC.

Transcriptional and post-translational modifications of B-Raf in quinol-thioether induced tuberous sclerosis renal cell carcinoma.

Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2(EK/+) ), bearing a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) gene, with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. These tumors have increased protein expression of B-Raf, C-Raf (Raf-1), and increased expression and activity of ERK kinase. Similar changes are observed in Raf kinases following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), cells that are also null for tuberin. Herein, we utilized LC-MS/MS to identify constitutive phosphorylation of S345 and S483 in both 100- and 95-kDa forms of B-Raf in QTRRE cells. Using microRotofor liquid-phase isoelectric focusing, we identified four fractions of B-Raf that contain different post-translational modification profiles in QTRRE cells. Amplification of the kinase domain of B-Raf from QTRRE cells, outer-stripe of the outer medulla of 8-month TGHQ- or vehicle-treated Tsc-2(+/+) and Tsc-2(EK/+) rats, as well as tumors excised from 8-month TGHQ-treated Tsc-2(EK/+) rats revealed three splice variants of B-Raf within the kinase domain. These splice variants differed by approximately 340, 544, and 600 bp; confirmed by sequencing. No point mutations within the kinase domain of B-Raf were identified. In addition, B-Raf/Raf-1/14-3-3 complex formation in the QTRRE cells was decreased by sorafenib, with concomitant selective decreases in p-ERK levels. Transcriptional and post-translational characterization of critical kinases, such as B-Raf, may contribute to the progression of tuberous sclerosis RCC. (246/250) © 2015 Wiley Periodicals, Inc.

Probing a 3,4'-bis-guanidinium diaryl derivative as an allosteric inhibitor of the Ras pathway.

Mutations in the Ras-pathway occur in 40-45% of colorectal cancer patients and these are refractory to treatment with anti-EGFR-targeted therapies. With this in mind, we have studied novel guanidinium-based compounds with demonstrated ability to inhibit protein kinases. We have performed docking studies with several proteins involved in the Ras-pathway and evaluated 3,4'-bis-guanidinium derivatives as inhibitors of B-Raf. Compound 3, the most potent in this series, demonstrated strong cytotoxicity in (WT)B-Raf colorectal cancer cells and also cells with (V600E)B-Raf mutations. Cell death was induced by apoptosis, detected by cleavage of PARP. Compound 3 also potently inhibited ERK1/2 signalling, inhibited EGFR activation, as well as Src, STAT3 and AKT phosphorylation. Mechanistically, compound 3 did not inhibit ATP binding to B-Raf, but direct assay of B-Raf activity was inhibited in vitro. Summarizing, we have identified a novel B-Raf type-III inhibitor that exhibits potent cellular cytotoxicity.

Abstract 4692: BGB-283, a novel RAF kinase and EGFR dual inhibitor, displays potent antitumor activity in B-RAF mutated colorectal cancers

Abstract Oncogenic B-RAF, which drives cell transformation and proliferation, has been detected in approximately 70% of human malignant melanomas and 5-15% of colorectal cancers (CRC). B-RAFV600E mutation, which gives rise to constitutive MAPK signaling, accounts for at least 90% of oncogenic B-RAF mutations. Despite the remarkable clinical activities achieved by vemurafenib and dabrafenib in treating B-RAFV600E metastatic melanoma, their clinical efficacy in B-RAFV600E CRC is far less impressive. Prior studies suggested that feedback activation of EGFR and MAPK signaling upon B-RAF inhibition contributed to the intrinsic resistance of CRC to the first generation B-RAF inhibitors. This report represents the first characterization of a dual RAF kinase/EGFR inhibitor, BGB-283, which is currently under clinical investigations. BGB-283 is a potent and selective pan-RAF and EGFR inhibitor with IC50 ranging from 5 to 47 nM on RAF and EGFR kinases. In vitro, BGB-283 potently inhibits B-RAFV600E-activated ERK phosphorylation and cell proliferation. It demonstrates selective cytotoxicity and preferentially inhibits proliferation of cancer cells harbouring B-RAFV600E and EGFR mutation/amplification. In B-RAFV600E CRC cell lines, BGB-283 effectively inhibits the reactivation of EGFR and achieved sustained inhibition of MAPK pathway. In vivo, BGB-283 is highly efficacious in inhibiting tumor growth accompanied by partial and complete tumor regressions in both BRAFV600E mutant cell derived CRC xenograft models, including HT29, Colo205, WiDr, as well as two primary human CRC xenograft models. In particular, BGB-283 shows compelling efficacy and potent inhibition of EGFR/MAPK signaling in WiDr xenograft model where EGFR reactivation occurs upon B-RAF inhibition. These findings support BGB-283 as a potent antitumor drug candidate with clinical potential for treating CRC harboring B-RAFV600E mutation. Citation Format: Zhiyu Tang, Xi Yuan, Rong Du, Shing-Hu Cheung, Guoliang Zhang, Jing Wei, Yuan Zhao, Yingcai Feng, Yi Zhang, Yunguang Du, Xiaoxia Hu, Wenfeng Gong, Yong Liu, Yajuan Gao, Rui Hao, Jiafu Ji, Lianhai Zhang, Shuangxi Li, David Sutton, Min Wei, Changyou Zhou, Lai Wang, Lusong Luo. BGB-283, a novel RAF kinase and EGFR dual inhibitor, displays potent antitumor activity in B-RAF mutated colorectal cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4692. doi:10.1158/1538-7445.AM2015-4692

Abstract 477: Inhibition of MEK confers hypersensitivity to X-radiation in the context of BRAF mutation in a model of childhood astrocytoma

Abstract Patients with recurrent low-grade brain tumors or anaplastic astrocytoma may receive radiation treatment, however, the long-term sequellae from radiation treatment can be severe. For pediatric astrocytomas, low-grade tumors are associated with activation of BRAF through a tandem duplication that results in the KIAA1549-BRAF fusion (Grade 1) or through an activating point mutation of BRAF (predominantly V600E; Grade 2-4) and is the most common lesion in low- and intermediate-grade astrocytoma. Previous studies have indicated synergy between MEK inhibition and ionizing radiation therapy (XRT), although the proposed mechanisms for synergy differ. We have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic childhood astrocytoma. The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant (V600E) glioblastoma cell lines (DBTRG-05MG, AM380c1). The effect of MEK inhibition (selumetinib, AZD6244, ARRY-142886), XRT (2 Gy daily fractionated doses), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. Inhibition of MEK signaling by selumetinib, suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of TORC1 signaling and decreased levels of FANCD2 protein, whereas inhibition of TORC1 signaling was less in DBTRG-05MG cells and even less in AM380c1 cells. The level of TORC1 inhibition by selumetinib was associated with loss of clonogenic survival, and suppression of FANCD2 in vitro. In vivo we examined the sensitivity of BT-40 xenografts to selumetinib (75 mg/kg BID x 42 days, PO), XRT (10 Gy delivered as 2 Gy daily fractions), or selumetinib combined with XRT. For control tumors, median time to event was 17 days, whereas for XRT alone it was 80 days (P<0.0001). Selumetinib treatment induced complete regression of most tumors with progression starting around week 5 of the 6-week treatment period, consistent with previous data, with median time to event being 51 days (P<0.0001 vs control). In contrast to XRT or selumetinib as single agents, the combination caused complete regression of all tumors, with only 4 of 10 (40%) recurrent tumors within the 24 weeks observation period, and significantly better than radiation or selumetinib alone (P<0.0001 for both comparisons). These data add to studies that indicate synergy between XRT and MEK inhibitors, and suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF. Combining MEK inhibition with XRT may allow maintenance of tumor control at lower doses of XRT that would decrease long-term sequellae for children with BRAF-mutant brain tumors. Citation Format: Adam W. Studebaker, Kathryn Bondra, Star Seum, Justin Leasure, Christopher Chronowski, Changxian Shen, Doris Phelps, Paul D. Smith, Raushan T. Kurmasheva, Xiaokui Mo, Peter J. Houghton. Inhibition of MEK confers hypersensitivity to X-radiation in the context of BRAF mutation in a model of childhood astrocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 477. doi:10.1158/1538-7445.AM2015-477

Abstract 2008: Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation in human cancer

Abstract Affinity purification-mass spectrometry (AP-MS) is a highly effective method of identifying and quantifying the individual constituents of molecular machines that are dynamically regulated by diverse cellular stimuli during tumor initiation, progression and metastasis. Seven In absentia homolog 2 (SIAH2) is an evolutionarily conserved E3 ubiquitin ligase and a key component of a molecular machine called “SIAH2-dependent proteolytic machinery” that serves as the most downstream signaling module critical for proper oncogenic ERBB/RAS signal transduction. As a newly identified promising anti-K-RAS and anticancer drug target, it is important to understand the dynamic regulation of SIAH function, substrate recognition and complex formation during tumor initiation, progression and metastasis. Our aim was to delineate the molecular regulation, substrate selection and target recognition of the SIAH proteolytic machinery in K-Ras-driven cellular transformation and oncogenesis; and to explore the SIAH-dependent K-RAS oncogene addiction and its future therapeutic application. In this study, affinity purification was conducted in triplicates to isolate the SIAH2 protein complexes from two panels of human epithelial cells: For the lung epithelial cells: we used the normal bronchial epithelial cells (BEAS-2B), RAS-transformed BEAS-2B (BZR) and non-small cell lung cancer (NSCLC) A549 cells with oncogenic K-RAS activation. For the mammary epithelial cells: we used normal mammary epithelial cells (HMEC and MCF-10A), benign breast tumor cells (MCF-7), MDA-MB-231 that carries both oncogenic K-RAS and B-RAF activation and MDA-MB-468. The extracted proteins were trypsin-digested and peptides subjected to high throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. SIAH2 was observed to propagate downstream RAS signals by affecting proteins involved in cellular junctions, focal adhesion, metabolism, mitochondrial function and kinase activities. Using Western blots, immunofluorescence staining and co-immunoprecipitation assays, we have verified and confirmed that focal adhesion proteins, TRIP6 (Thyroid Receptor Interacting Protein 6) and FHL2 (Four and Half Lim domain protein) are SIAH2 substrates in human cancer cell lines lung cancer (A549) and triple negative breast cancer (MDA-MB-231) and breast cancer (MDA-MB-468). We found that TRIP6 primarily localizes to the focal adhesions, cell junctions and the nucleus of the cells. Overexpression of SIAH2 induces the degradation of exogenous and endogenous TRIP6 while Inhibition of SIAH2 enzymatic function results in a stabilization of TRIP6 and mis-localization of TRIP6 in human cancer cells. In conclusion, we report that SIAH2 regulates focal adhesion, cell junction and cellular attachment by down regulating TRIP6 expression levels and may induce a more motile and aggressive phenotype in human cancer cells. Citation Format: Monicah M. Njogu, Ming Lei Bian, Jamie Eisner, Amy H. Tang. Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation in human cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2008. doi:10.1158/1538-7445.AM2015-2008