Introduction

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 61/904,623, filed November 15, 2013, the content of which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grant numbers P30RR032136, CA009658, AI007363, GM00874, R01 AR-26599, CA77267, R01 CA120777, R01 CA134799 and GM103415 awarded by the National Institutes of Health. The government has certain rights in the invention.

Background

[0003] Rates of melanoma are steadily on the rise, with a current lifetime incidence of 1 in 50 individuals (Balch, et al . (2004) CA: A Cancer Journal for Clinicians 54 (3) : 131-49; Smalley, et al . (2005) Anticancer Therapy 5 (6) : 1069-780. In over 65% of human malignant melanomas, the BRAF oncogene encodes BRAF ^V600E (Davies, et al . (2002) Nature 417 (6892 ) : 949-54 ) , a protein whose expression increases melanoma progression via activation of signaling cascades and downstream genes (Koya, et al . (2012) Cancer Res. 72 (16) :3928-37; Joseph, et al . (2010) Proc . Natl. Acad. Sci. USA 107 (33 ) : 14903-8 ; Yang, et al . (2010) Cancer Res. 70 (13) : 5518-27; Vultur, et al . (2011) Clin. Cancer Res. 17 (7) : 1658-63 ; Chapman, et al (2011) New Engl . J. Med. 364 (26) : 2507-16) . Vemurafenib, also known as PLX4032, is a small molecule inhibitor that binds to the ATP-binding site of mutated BRAF kinase, inhibiting extracellular signal - regulated kinase (ERK) signaling only in tumor cells expressing BRAF ^veo0E (Joseph, et al . (2010) supra) . Clinical use of PLX4032, to treat BRAF ^V600E tumors, induces rapid tumor regression and has revolutionized targeted therapy for melanoma (Boni, et al . (2010) Cancer Res. 70 (13) :5213- 9; Khalili, et al . (2012) Clin. Cancer Res. 18 (19) : 5329-40 ; Koya, et al . (2012) supra; Straussman, et al (2012) Nature 487 (7408) : 500-4) . However, resistance to PLX4032 typically develops within one year (Vultur, et al . (2011) supra; Joseph, et al . (2010) supra; Straussman, et al (2012) supra) , underscoring the need for increased understanding of molecular mechanisms underlying BRAF ^V600E and for developing additional therapeutic strategies.

[ 0004 ] The use of human melanoma cell lines is well established for in vitro and in vivo investigations of BRAF ^V500E melanoma (Herlyn & Fukunaga-Kalabis (2010) J ^" . Invest. Dermatol. 130 (4 ) : 911-2 ; S0ndergaard et al . (2010) J ^" . Translation. Med. 8:39) . However, human cell lines rely on xenografts in immune -compromised mice, which limit studies of host-tumor cell interactions in a genetically identical (syngeneic) background. Mouse models of BRaf ^v600E malignant melanoma have provided researchers with the ability to investigate in vivo tumor responses to PLX4032 treatment (Dankort, et al (2009) Nature Genetics 41(5) :544- 52; Dhomen, et al (2009) Cancer Cell 15 (4) : 294-303. Backcrossing these mice onto a pure background has allowed for in vivo testing of immunologic therapies in a syngeneic system (Hooijkaas, et al . (2012) Oncoimmunology 1(5) :609- 617; Hooijkaas, et al . (2012) Am. J. Pathol. 181(3) :785- 94) . However, for in vitro analysis of the molecular mechanisms underlying BRaf ^V600E melanoma, stable cell lines that recapitulate the human disease are needed. These lines could be used to identify critical mechanisms: signal transduction pathways, patterns of gene expression, and interactions with cells within the tumor microenvironment (Herlyn & Fukunaga-Kalabis (2010) supra) . [0005] The isolation of murine BRaf ^V600E cell lines from mouse models of BRaf ^v600E melanoma has been reported (Koya, et al . (2012) supra; Hooijkaas, et al . (2012) supra) . However, given the heterogeneity of human BRAF ^V600E cell lines, the establishment of only two murine cell lines may not adequately represent the true biology of BRaf ^V600E cells in vitro. For example, both of the reported murine BRaf ^v600E cell lines are only partially sensitive to PLX4032 and PLX4720 (Koya, et al . (2012) supra; Hooijkaas, et al .

(2012) supra; Knight, et al . (2013) J ^" . Clin. Invest. 123 (3) : 1371-81) , with respect to decreased pERK signaling and cellular proliferation. These in vitro results differ from in vivo results seen in a corresponding mouse model

(Hooijkaas, et al . (2012) supra) and with human BRAF ^V600E cell lines (Joseph, et al . (2010) supra; Yang, et al .

(2010) supra; Straussman, et al (2012) supra) .

[0006] Therefore, there is a need in the art for a transplantable tumor model, based on cells from a defined genetic background, which would enable more rapid and reproducible evaluations of tumor growth in multiple tissue compartments as well as a better understanding of the molecular mechanism underlying disease progression and resistance .

Summary of the Invention

[0007] This invention is a method for producing a BRAF ^V600E mouse melanoma cell line. The method involves the steps of

(a) excising a first melanoma tumor from a mouse, wherein

V600E cells of the tumor carry a Cre-activated allele of BRAF ;

(b) dissociating cells of the excised tumor, (c) injecting the dissociated tumor cells into an immune -compromised mouse, without culturing the dissociated tumor cells, (d) allowing the injected tumor cells to develop into a second tumor, (e) excising the second tumor from the mouse, and (f) culturing the excised tumor cells to produce a BRAF ^V600E melanoma cell line. A BRAF ^V600E mouse melanoma cell line produced by the method is also provided.

Detailed Description of the Invention

[0008] Mouse models of BRAF ^V600E provide a system for studying melanoma in vivo. Accordingly, melanoma cell lines have now been established from the conditional mouse model of metastatic melanoma, Tyr : : CreER; Braf ^CA ; Pten ^{lox4~5/lox4'5} . The melanoma cell lines grow readily in culture, are sensitive to PLX4032, and are transplantable in syngeneic hosts. Given that the cells of the invention recapitulate human disease, they provide a valuable model for studying the molecular mechanisms of malignant melanoma in vitro, as well as a transplantable model of melanoma progression in vivo .

[0009] Accordingly, the present invention is a method for producing a BRAF ^V600K mouse melanoma cell line. The method of the invention comprises or consists of the steps of excising a first melanoma tumor from a mouse, wherein cells of the tumor carry a Cre-activated allele of BRAF ^V600E ; dissociating cells of the excised tumor; injecting the dissociated tumor cells into an immune -compromised mouse, without culturing the dissociated tumor cells; allowing the injected tumor cells to develop into a second tumor; excising the second tumor from the mouse; and culturing the excised tumor cells to produce a BRAF ^V600E melanoma cell line .

[0010] As is known in the art, melanoma is a malignant tumor arising from melanocytes in the skin or other sites, and which may contain dark pigment. In certain embodiments, the cells of the melanoma cell line of this invention have a mutation in the v-raf murine sarcoma viral oncogenes homolog Bl (BRAF) isoform of the RAF kinase. Ninety percent of braf mutations are accounted for by a thymine to adenine single-base change at position 1,799. This missense mutation, located in exon 15, results in a change at residue 600 of the BRAF protein that substitutes glutamine for valine (V600E; Davies, et al . (2002) Nature 417:9490- 54) . BRAF ^VS00E can gain 500 -fold increased activation, stimulating the constitutive activation of MEK/ERK signaling in tumor cells. Furthermore, it allows activation of this signaling cascade in the absence of any extracellular stimuli, allowing the cell to become self- sufficient in growth signals within this pathway. Accordingly, in particular embodiments, the cells of the melanoma cell line carry the BRAF ^V600E mutation.

[ 0011 ] Mutation of BRAF can be carried out by conventional methods. In particular embodiments, the mutation is carried out using the Cre-Lox system to create a conditionally active (CA) mutant. By way of illustration, DNA encompassing mouse BRaf exons 14-16 can be modified such that exon 16 is replaced with a HSV-thymidine kinase cassette. A LoxP-flanked cassette containing the 3' 382 base pairs (bp) of intron 14, the human BRAF cDNA containing exons 15-18, the mouse BRaf polyadenylation sequences, and a PGK-neo cassette can be inserted into intron 14 upstream of a modified exon 15 that encodes BRafV600E. The LoxP-flanked cassette can be introduced into mouse embryonic stem cells by conventional methods. Subsequently, embryonic stem cell clones can be screened to identify targeting of BRAF. Clones with a single copy integration of the mutant BRAF can be injected into C57B1/6 blastocysts and germline transmission assessed by, e.g., PCR. Chimeric mice can be crossed to yield homozygous (BRaf ^CA/CA ) or heterozygous (BRaf ^CA/+ ) mice. See, e.g., Dankort, et al . (2007) Genes Dev. 21:379-84. Furthermore, BRAF ^CA mice can be backcrossed, e.g., to C57B1/6 mice, to achieve a pure background {e.g., less than 5%, 4%, 3%, 2% or 1% of the genome is from another mouse line) .

[ 0012 ] Resulting BRaf ^CA mice harbor the germ- line conditional BRaf ^veoOE allele, the expression of which can be initiated at physiological levels under the control of the gene's chromosomal promoter by the action of Cre recombinase . When the BRaf ^CA allele is combined with a conditionally active Cre recombinase, e.g., CreER ^T2 that is specifically expressed in melanocytes (Bosenberg, et al . (2006) Genesis 44:262-7) and exposed to 4 -hydroxytamoxifen (4-HT) , highly pigmented lesions appear by 21-28 days after 4-HT administration. As is seen in human compound melanocytic nevi , BRaf ^v600E - induced pigmented lesions of the transgenic mouse are frequently located at the dermal /epidermal junction with extension into the dermis. See Dankort, et al . (2009) Nat. Genet. 41:544-552.

[ 0013 ] As is known in the art BRAF ^V600E elicits sustained activation of the BRAF→ EK1 /2→ERK1/2 MAP kinase pathway. While contributing to the aberrant pathophysiological characteristics of the melanoma cell (Wellbrock, et al . (2004) Nat. Rev. Mol . Cell Biol. 5:875-85; Mercer & Pritchard (2003) Biochim. Biophys . Acta 1653:25-40), progression to malignant melanoma is often accompanied by silencing of one or more tumor suppressor genes, most commonly PTEN (phosphatase and tensin homolog) , INK4A (cyclin-dependent kinase 4 inhibitor A) and/or ARF (alternate reading frame) (Garraway, et al . (2005) Nature 436:117-22; Wellbrock, et al . (2004) Cancer Res. 64:2338-4; Chin, et al . (2006) Genes Dev. 20:2149-82; Lin, et al . (2008) Cancer Res. 68:664-73) . Thus, in particular embodiments of this invention, the tumors of the instant method are malignant and, in addition to carrying a Cre- activated allele of BRAF ^V600E , said tumors are deficient in one or more tumor suppressor genes. In particular embodiments, the tumors are deficient in one or more of PTEN, INK4A or ARF. A tumor deficient in a tumor suppressor gene is intended to mean that the cells does not express the tumor suppressor gene, e.g., as the result of a gene knockout, gene disruption, or siRNA-mediated gene silencing .

[ 0014 ] As a first step in the method of the invention, a melanoma tumor of a mouse carrying a Cre-activated allele of BRAF ^VS00E is excised, e.g., by excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy. An "excisional biopsy" refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An "incisional biopsy" refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor. A "needle biopsy" generally contains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison' s Principles of Internal Medicine, Kasper, et al . , eds . , 16th ed. , 2005, Chapter 70, and throughout Part V.

[ 0015 ] Once excised, the BRAF ^600E cells of the tumor are dissociated, e.g., by enzymatic dissociation with collagenase and DNase, by mechanical dissociation in a blender, by teasing with tweezers, using mortar and pestle, cutting into small pieces using a scalpel blade, and the like, to yield single, isolated cells. In some embodiments, the isolated primary tumor cells are briefly cultured

(e.g., 24 to 72 hours) in culture medium (e.g., Dulbecco's Modified Eagle Medium (DMEM) , DMEM/F-12 advanced medium, Roswell Park Memorial Institute (RPMI) medium, and the like) with animal serum (e.g., fetal bovine serum, human serum or an appropriate substitute thereof) to ensure and/or maintain viability. In other embodiments, the isolated primary tumor cells are directly injected into an immune -compromised mouse without culturing the dissociated tumor cells. Examples of immune -compromised mice of use in the invention include, but are not limited to, nonobese diabetic (NOD) /severe combined immunodeficient (SCID) mice, NOD/SCID/γ chain™ ¹¹ (NOG, NSG or DKO) mice and Nu/Nu (Nude) mice .

[0016] Upon introduction into the immune -compromised mouse, the tumor cells are allowed to develop into a second tumor, e.g., a tumor in the range of 5 to 20 mm in diameter, or more preferably 10 to 15 mm in diameter. The secondary tumor is subsequently excised, dissociated, and cultured in a suitable culture medium as described herein. Cells obtained by this method of the invention express high constitutive phosphorylation of ERK, are inhibited by Vemurafenib (PLX4032) , are readily transplantable into mice, and show no difference in growth rates in either NSG or syngeneic C57B1/6 mice.

[0017] BRAF-mutated tumors have been correlated with poor response to traditional chemotherapy and poor prognosis in melanoma, thyroid, and colon cancers (Tang & Lee (2010) J. Chin. Med. Assoc. 73:113-28; Houben, et al . (2004) J. Carcinog. 3:6) . Targeted therapies are of great interest for these cancer types and the elucidation of the structure and functions of the BRAF ^■ kinase is the subject of ongoing research. The approach of targeting oncogenic kinases has been successful in the treatment of cancers, with activating mutations in the kinase gene that drives their progression (Sawyers (2004) Nature 432:294-7) . For example-, Imatinib has been used in the treatment of chronic myeloid leukemia, which is driven by a characteristic mutant fusion protein Bcr-Abl, resulting in constitutive activation of the Abl kinase. Imatinib effectively blocks the Abl kinase producing an effective clinical response that can be correlated to the presence of the activating kinase mutation .

[ 0018 ] Similarly, BRAF- specific inhibitors such as GDC-0879 and PLX4720 effectively block BRAF ^V600E and thus block BRAF ^V600E - induced ERK activation. PLX4720 was designed according to the atomic structure of BRAF ^V600E and as a result has a 10-fold increased potency for BRAF ^V600E over the wild-type kinase (Tsai, et al . (2008) Proc . Natl. Acad. Sci. USA 105:3041-6) . These first -generation BRAF inhibitors were found to be effective in preclinical trials but underwhelmed in clinical trials. The emergence of second-generation inhibitors, such as the PLX4720 analogue PLX4032, is proving much better in clinical trials. The details of a phase I trial for PLX4032 announced an 80% response rate (as determined by complete or partial tumor regression) among patients with BRAF ^V600E mutations whereas patients without the mutation did not respond (Flaherty, et al . (2010) N. Engl. J. Med. 363:809-19) . This analysis provides proof of concept for the identification of other small BRAF ^veo0E inhibitors.

[ 0019 ] In this respect, the BRAF ^V600E melanoma cell line of this invention is of use in correlating in vitro studies on molecular mechanisms of melanoma with in vivo investigations on pathology, immunogenicity, and signaling in response to Vemurafenib or other targeted signal transduction inhibitors. In addition, the cell line of the invention will allow the in vivo induction of tumors in syngeneic or immune-compromised mice for the evaluation of targeted or combinatorial therapy in a well-controlled tumor model. Thus, agents that inhibit melanoma formation and may therefore be used in prophylactic treatment regimens or in the therapy of existing melanoma can be identified .

[ 00203 The following are provided as specific embodiments of the present invention. Other modifications of this invention will be readily apparent to those skilled in the art, without departing from the scope of this invention.

Example 1 : Murine BRaf ^v600E Cell Lines

[ 0021 ] Mouse BRaf ^v600E cell lines were produced by backcrossing the transgenic mouse model,

Tyr::CreER;Braf ^CA ;Pten ^lox/lox (Dankort, et al . (2009) supra) to a C57BL/6 (B6) background to achieve <98% non-B6 DNA; these mice are hereafter referred to as Braf/Pten mice. Initially, the establishment of stable cell lines from this model was difficult likely due to the heterogeneous population of cells in the primary tumor and the slow growth of the tumor cells in culture. Accordingly, a technique was developed, which was used to establish several stable cell lines that represent the in vivo biology of tumors in Braf/Pten mice.

[ 0022 ] It was found that the establishment of stable cell lines from Braf/Pten mice required a protocol of sequential in vivo and in vitro growth. Induced tumors (10 mm in diameter) were resected from Braf/Pten mice and dissociated into single cells by collagenase digestion according to established methods. It was observed that DMEM advanced/5% FBS media was optimal for survival of cells isolated from the primary tumor. However, these cells often grew slowing in culture, making them unsuitable for in vitro studies. To verify the tumorigenicity of dissociated primary tumor cells and cultured primary tumor cells, the cells were directly injected into NOD/SCID/γ chain ¹¹ " ¹¹ (NSG) mice, an immune -compromised, permissive host. When secondary tumors from the host mice reached 10-12 mm in diameter, they were surgically excised and cultured using the same dissociation protocol and culture media. Of the seven tumors that were processed with this sequence, five stable D4M (Dartmouth Murine Mutant Malignant Melanoma) cell lines were successfully developed from both male and female Braf/Pten mice, demonstrating that cell lines with this protocol can routinely be established.

Example 2: Characterization of D4 Cell Lines

[0023] Since both human BRAF ^V600E and mouse BRaf ^V600E tumors have constitutive activation of MAPK signaling cascades, levels of downstream pERK was evaluated in three D4M cell lines, 3A, 5A, and 7A. In keeping with a mutational activation of BRaf (Dankort, et al . (2009) supra) , D4M cell lines had high constitutive levels of pERK compared to B16F1 cells, which are Braf ^wt . Two D4M cell lines, D4M.3A

(from a male donor) and D4M.7A (from a female donor), were used for subsequent studies. Although these cell lines were generated using different modifications of the protocol described herein (3A, with culture before re-injection, 7A, without culture before re-injection) , they were morphologically similar in culture. All D4M cell lines generated were unpigmented, which is also observed in human melanoma cell lines such as VMM5 and VMM12 cells

(Blackburn, et al . (2007) Cancer Res. 67:10849-58; Blackburn, et al . (2009) Oncogene 28:4237-4248; Croteau, et al. (2012) J ^" . Cell. Physiol. 228:773-780; Huntington, et al. (2004) J ^" . Biol. Chem. 279:33168-176; Yamshchikov, et al . (2005) J. Immunol. 174:6863-71) . Further, both D4M.3A and D4M.7A grew in advanced DMEM/F-12 media with or without serum. Survival and growth in serum- free conditions, though significantly less than in serum-containing conditions (P>0.05), allows for analysis of secreted factors released from these cell lines in vitro.

[ 0024 ] PLX4032 binds V600E mutant BRAF with high affinity and inhibits ERK signaling output. Thus, the sensitivity of cultured D4M cells to this inhibitor was tested by first evaluating cell signaling after treatment with PLX4032, using DMSO-treated cells as negative controls and U0126- treated cells as positive controls. U0126 is a selective inhibitor of MEK1 and MEK2 , blocking downstream signals including phosphorylation of ERK. Treatment of D4M.3A, D4M.5A and D4M.7A cells with 3 μΜ PLX4032 for 45 or 90 minutes, led to decreased pERK, with no change in levels of pAKT. Treatment with U0126 decreased pERK levels after 10 minutes. VMM5 and VMM12 cells, human BRAF ^V600E cell lines of malignant melanoma (Huntington, et al . (2004) J. Biol. Che . 279 (32) : 33168-76) , showed similar responses to PLX4032 and U0126, with decreased pERK levels at 45 and 10 minutes respectively, and no change in pAK . D4M cell lines were, thus, comparable to human BRAF ^VS00E cell lines in their cell signaling response to PLX4032 treatment (Joseph, et al . (2010) supra; Yang, et al . (2010) supra; Straussman, et al (2012) supra) . This analysis indicated that D4M cell lines have characteristics similar to human BRAF ^V600E melanoma and BRaf ^V600E mouse models.

[ 0025 ] Cellular proliferation of D4M cell lines in response to PLX4032 was subsequently evaluated. Treatment with 3 μΜ PLX4032 significantly (P < 0.05) arrested in vitro growth of D4M.3A and D4M.7A cells over 2-3 days. Human tumors with BRAF ^V600E are dependent on constitutive activation of ERK signaling for cellular proliferation; thus, addition of PLX4032 causes growth arrest of BRAF ^V600E tumor cells in vitro (Joseph, et al . (2010) supra) . In keeping with reports of human BRAP ^VSOOE cell lines (Joseph, et al . (2010) supra; Yang, et al . (2010) supra), the response of the D4M cells to PLX4032 appeared to be primarily cytostatic. Previously published murine BRaf ^v600E cell lines are relatively resistant to PLX4032 and PLX4720 treatment compared to human BRAF ^V600E cell lines, with respect to decreased pERK signaling and cellular proliferation (Koya, et al . (2012) supra; Hooijkaas, et al . (2012) supra; Knight, et al . (2013) supra) .

[0026] To assess the tumorigenicity of D4 melanoma cells, a range (300-300,000) of cells were intradermally injected into B6 and NSG host mice. Each concentration of cells was injected into four B6 and four NSG mice. Of the eight mice injected with 300 cells, no tumors developed in any of the mice after 27 weeks. In contrast, it took only two weeks for tumors to be observed in mice that received >3,000 cells, and only one week for tumors to be observed in mice that received 300,000 cells (Table 1) . Tumors arose with similar kinetics in B6 and NSG host mice; there was no significant difference in the average tumor size between NSG and B6 mice injected with 3,000 D4M.3A cells, at any time point. Histologic examination revealed no gross morphologic differences between tumors in B6 or NSG hosts. It was concluded that the D4M cells were equally tumorigenic in both immune-compromised and syngeneic hosts. TABLE 1

Bold values indicate when 100% of mice developed tumors.

[0027] PLX4720 (a compound similar to PLX4032; Tsai, et al .

(2008) Proc. Natl. Acad. Sci USA 105:3041-46) was also tested for efficacy in inhibiting tumor progression and phosphorylated ERK signaling in vivo. D4M.3A cells were injected intradermally into B6 mice (300,000 cells/mouse) . When tumors reached 5 mm in diameter (8 days post- injection) , mice were either given chow containing PLX4720

(Plexxikon; Berkeley, CA) or control chow. Tumors were measured 4 and 7 days after the start of treatment and tumor volumes were calculated. After 7 days of treatment, mice that were fed the PLX4720 chow had significantly smaller tumors than mice that were fed the control chow

(P<0.05) . Mice injected with D4M.7A cells showed comparable results, with smaller tumors after 7 days in mice fed the PLX4720 chow compared with those fed the control chow. In addition, tumors in mice fed the PLX4720 chow for 7 days had decreased pERK levels compared to tumors in mice fed the control chow.

[0028] To further verify that D4M cell lines have melanoma characteristics, expression of the murine melanoma marker, pmel, was examined. Several melanocyte differentiation antigens (MDAs) , such as gplOO (also referred to as Pmel) are widely expressed in human melanoma and recognized by tumor- infiltrating lymphocytes (Kawakami (1994) Proc . Natl. Acad. Sci. USA 91 (14) : 6458-6462) , making it a potentially useful biomarker. Relatively low levels of pmel expression were observed in D4M.3A cultured cells, however, expression increased in tumors that arose when D4M.3A cells were injected into either NSG or B6 mice. Further, when these tumors were excised and re-cultured once or twice, pmel levels fell in culture, but increased again upon implantation into mice (P<0.0005) . This trend was also observed with Mart-1, tyrosinase and Tryp-1 expression. This indicates that D4M cells maintain the ability to express pmel, in vivo and in vitro. Further, the in vivo upregulation of pmel indicates that D4M cells are responding to host factors within the tumor microenvironment . The fact that equal levels of increase were seen upon transplantation of the D4M cells into either NSG or B6 mice indicates that non- immune cells may be mediating most of this increase. Indeed, the stromal compartment of adjacent fibroblasts has been implicated as one prominent modulator of tumor cell behavior in vivo (Bhowmick, et al . (2004) Nature 432 (7015) : 332-7 ; Smalley, et al. (2005) Exp. Rev. Anticane . Ther. 5 (6) : 1069-78 ; Eck, et al. (2009) J ^" . Autoimmun. 33 (3 -4 ) : 214-21 ; Marsh, et al . (2013) Biochim. Biophys . Acta 1832 (7) : 1070-8) . In addition, discrepancies in the levels of gene expression between cultured human melanoma cells and those excised from nude mice (Croteau, et al . (2012) J ^" . Cell. Physiol. 228:773-780) have been found, underscoring the importance of utilizing both in vitro and in vivo model systems.

[ 0029 ] Investigations with human melanoma cell lines and clinical tissue samples demonstrate that PLX4720 (a precursor of PLX4032) treatment leads to increased expression of MDAs such as Pmel (Boni, et al . (2010) Cancer Res. 70:5213-9; Frederick, et al . (2013) Clin. Cancer Res. 19:1225-31) . Since D4M cells were sensitive to PLX4032, cells were treated with PLX4032 and mRNA levels of pmel were analyzed. In D4M.3A and D4M.7A cell lines, basal levels of pmel were relatively low and comparable to levels in 3T3 cells. However, an approximate 4-fold increase (P>0.05) in pmel expression was observed 48 hours after the addition of PLX4032. Similar increases were observed in two D4M cell lines but not in Braf ^wt 3T3 cells. Similarly, when the two human melanoma cell lines (VMM5 and VMM12) were treated with PLx4032 for 48 hours, pmel levels significantly increased, while no effect was observed in human fibroblasts.

[ 003 0 ] For functional analysis of Pmel antigen recognition, a cytotoxicity assay was used to assess melanoma cell sensitivity to lysis by Pmel TCR transgenic T cells

(Overwijk, et al . (2003) J. Exp. Med. 198:569-80) . Activated Pmel T cells were added to DMSO- or PLX4032- treated D4M.3A cells at different E:T ratios. PLX4032 treatment increased lysis of D4M .3A cells by Pmel T cells compared to DMSO treatment. EL-4 (antigen-negative) cells either pulsed or unpulsed with Pmel peptide were used as control targets, and Pmel T cells specifically recognized and lysed peptide-pulsed, but not unpulsed, EL-4 cells. Taken together, these data are consistent with increased MDA expression in human melanoma cell lines in response to BRAF ^V600E inhibition (Boni, et al . (2010) supra), and further validate the D4M cell lines as bone fide melanoma cells and an appropriate murine model system. Although D4M cell lines do not have high endogenous levels of MDAs in vitro, the data herein illustrate that D4M.3A cells express Pmel in a functionally-relevant manner. [ 003 1 ] D4M cell lines recapitulated human BRAF ^V600E melanoma in vitro. However, unlike human cell line xenografts, D4M cell lines were readily transplantable into syngeneic host mice, which allows for immunological studies. The fact that there was no significant difference between tumorigenicity and tumor volume in NSG and B6 hosts indicates that D4M cell lines are relatively non- immunogenic , like murine B16 cells (Leveson, et al . (1979) Cancer Res. 39:582-6; Ashley & Kotlarski (1986) Cell. Immunol. 101:156-67) . This is consistent with the expression of low levels of MDAs in culture and the relative paucity of immune cells in the tumor tissue (as seen by histology) .

[ 0032 ] Given the heterogeneity of human BRAF ^V600E melanoma cell lines, having multiple murine BRaf ^v600E cell lines is an important resource for the scientific community. The established BRaf ^v600E SMI cell line has been a useful model in supporting the therapeutic potential of combining BRAF inhibitors with immunotherapy (Knight, et al . (2013) supra) . D4M cells are expected to be similarly useful in developing models of new therapeutic modalities. In addition, the fact that, at 3 μΜ PLX4032, the D4M cells show substantial decreases in pERK and decrease in cell proliferation, indicates that these cells are distinct from those described in the art .