Model of the TVA receptor determinants required for efficient infection by subgroup A avian sarcoma and leukosis viruses.

UNLABELLED: The study of the interactions of subgroup A avian sarcoma and leucosis viruses [ASLV(A)] with the TVA receptor required to infect cells offers a powerful experimental model of retroviral entry. Several regions and specific residues in the TVA receptor have previously been identified to be critical determinants of the binding affinity with ASLV(A) envelope glycoproteins and to mediate efficient infection. Two homologs of the TVA receptor have been cloned: the original quail TVA receptor, which has been the basis for most of the initial characterization of the ASLV(A) TVA, and the chicken TVA receptor, which is 65% identical to the quail receptor overall but identical in the region thought to be critical for infection. Our previous work characterized three mutant ASLV(A) isolates that could efficiently bind and infect cells using the chicken TVA receptor homolog but not using the quail TVA receptor homolog, with the infectivity of one mutant virus being >500-fold less with the quail TVA receptor. The mutant viruses contained mutations in the hr1 region of the surface glycoprotein. Using chimeras of the quail and chicken TVA receptors, we have identified new residues of TVA critical for the binding affinity and entry of ASLV(A) using the mutant glycoproteins and viruses to probe the function of those residues. The quail TVA receptor required changes at residues 10, 14, and 31 of the corresponding chicken TVA residues to bind wild-type and mutant ASLV(A) glycoproteins with a high affinity and recover the ability to mediate efficient infection of cells. A model of the TVA determinants critical for interacting with ASLV(A) glycoproteins is proposed. IMPORTANCE: A detailed understanding of how retroviruses enter cells, evolve to use new receptors, and maintain efficient entry is crucial for identifying new targets for combating retrovirus infection and pathogenesis, as well as for developing new approaches for targeted gene delivery. Since all retroviruses share an envelope glycoprotein organization, they likely share a mechanism of receptor triggering to begin the entry process. Multiple, noncontiguous interaction determinants located in the receptor and the surface (SU) glycoprotein hypervariable domains are required for binding affinity and to restrict or broaden receptor usage. In this study, further mechanistic details of the entry process were elucidated by characterizing the ASLV(A) glycoprotein interactions with the TVA receptor required for entry. The ASLV(A) envelope glycoproteins are organized into functional domains that allow changes in receptor choice to occur by mutation and/or recombination while maintaining a critical level of receptor binding affinity and an ability to trigger glycoprotein conformational changes.

Transdermal Delivery of Ultradeformable Cationic Liposomes Complexed with miR211-5p (UCL-211) Stabilizes BRAFV600E+ Melanocytic Nevi.

Small non-coding RNAs (e.g. siRNA, miRNA) are involved in a variety of melanocyte-associated skin conditions and act as drivers for alterations in gene expression within melanocytes. These molecular changes can potentially affect the cellular stability of melanocytes and promote their oncogenic transformation. Thus, small RNAs can be considered as therapeutic targets for these conditions, however, their topical delivery to the melanocytes through the epidermal barrier is challenging. We synthesized and extensively evaluated ultradeformable cationic liposome (UCLs) carriers complexed with synthetic microRNAs (miR211-5p; UCL-211) for transdermal delivery to melanocytes. UCL-211 complexes were characterized for their physicochemical properties, encapsulation efficiency, and deformability, which revealed a significant advantage over conventional liposomal carriers. Increased expression of miR211-5p stabilizes melanocytic nevi and keeps them in growth-arrested state. We did a comprehensive assessment of cellular delivery, and biological activity of the miR211-5p carried by UCL-211 in vitro and their permeation through the epidermis of intact skin using ex vivo human skin tissue explants. We also demonstrated, in vivo , that topical delivery of miR211-5p by UCL-211 stabilized BRAFV600E+ nevi melanocytes in a benign nevi state.

Genetic Silencing of AKT Induces Melanoma Cell Death via mTOR Suppression.

Aberrant activation of the PI3K-AKT pathway is common in many cancers, including melanoma, and AKT1, 2 and 3 (AKT1-3) are bona fide oncoprotein kinases with well-validated downstream effectors. However, efforts to pharmacologically inhibit AKT have proven to be largely ineffective. In this study, we observed paradoxical effects following either pharmacologic or genetic inhibition of AKT1-3 in melanoma cells. Although pharmacological inhibition was without effect, genetic silencing of all three AKT paralogs significantly induced melanoma cell death through effects on mTOR. This phenotype was rescued by exogenous AKT1 expression in a kinase-dependent manner. Pharmacological inhibition of PI3K and mTOR with a novel dual inhibitor effectively suppressed melanoma cell proliferation in vitro and inhibited tumor growth in vivo. Furthermore, this single-agent-targeted therapy was well-tolerated in vivo and was effective against MAPK inhibitor-resistant patient-derived melanoma xenografts. These results suggest that inhibition of PI3K and mTOR with this novel dual inhibitor may represent a promising therapeutic strategy in this disease in both the first-line and MAPK inhibitor-resistant setting.

Prior Exposure to Coxsackievirus A21 Does Not Mitigate Oncolytic Therapeutic Efficacy.

Oncolytic viruses (OVs) are being developed as a type of immunotherapy and have demonstrated durable tumor responses and clinical efficacy. One such OV, Coxsackievirus A21 (CVA21), exhibited therapeutic efficacy in early phase clinical trials, demonstrating the ability to infect and kill cancer cells and stimulate anti-tumor immune responses. However, one of the major concerns in using this common cold virus as a therapeutic is the potential for innate and adaptive immune responses to mitigate the benefits of viral infection, particularly in individuals that have been exposed to coxsackievirus prior to treatment. In this study, we assess melanoma responses to CVA21 in the absence or presence of prior exposure to the virus. Melanomas were transplanted into naïve or CVA21-immunized C57BL6 mice and the mice were treated with intratumoral (IT) CVA21. We find that prior exposure to CVA21 does not dramatically affect tumor responses, nor does it alter overall survival. Our results suggest that prior exposure to coxsackievirus is not a critical determinant of patient selection for IT CVA21 interventions.

Aspirin Protects Melanocytes and Keratinocytes against UVB-Induced DNA Damage In Vivo.

UVR promotes skin cancer through multiple mechanisms, including induction of inflammation, oxidative stress, and DNA damage such as 8-oxoguanine and cyclobutane pyrimidine dimers. We investigated whether the anti-inflammatory activities of aspirin (acetylsalicylic acid [ASA]) could protect against UVB-induced DNA damage and skin carcinogenesis. ASA reduced UVB-induced 8-oxoguanine and cyclobutane pyrimidine dimers in Melan-A melanocytes and HaCaT keratinocytes. Skin from UVB-irradiated C57BL/6 mice receiving 0.4 mg ASA daily by gavage exhibited less inflammation, fewer sunburn cells, and reduced 8-oxoguanine lesions than skin from irradiated control animals. ASA similarly reduced UVB-induced sunburn cells, 8-oxoguanine, and cyclobutane pyrimidine dimer lesions in skin of melanoma-prone TN61R mice, and this was associated with decreased prostaglandin E2 in plasma and skin. These effects of ASA, however, did not delay melanoma onset in TN61R mice exposed to a single neonatal dose of UVB. In SKH1-E mice prone to squamous cell carcinoma, ASA reduced plasma and skin prostaglandin E2 levels and indices of UVB-induced DNA damage and delayed squamous cell carcinoma onset induced by chronic UVB. These results indicate that ASA can protect against UVB-induced inflammation in skin and reduce UVB-induced DNA damage in both melanocytes and keratinocytes. These effects translated into greater chemopreventive efficacy for UVB-induced squamous cell carcinoma than melanoma mouse models.

BRAF inhibition in melanoma is associated with the dysregulation of histone methylation and histone methyltransferases.

The development of mutant BRAF inhibitors has improved the outcome for melanoma patients with BRAFV600E mutations. Although the initial response to these inhibitors can be dramatic, sometimes resulting in complete tumor regression, the majority of melanomas become resistant. To study resistance to BRAF inhibition, we developed a novel mouse model of melanoma using a tetracycline/doxycycline-regulated system that permits control of mutant BRAF expression. Treatment with doxycycline leads to loss of mutant BRAF expression and tumor regression, but tumors recur after a prolonged period of response to treatment. Vemurafenib, encorafenib and dabrafenib induce cell cycle arrest and apoptosis in BRAF melanoma cell lines; however, a residual population of tumor cells survive. Comparing gene expression in human cell lines and mouse tumors can assist with the identification of novel mechanisms of resistance. Accordingly, we conducted RNA sequencing analysis and immunoblotting on untreated and doxycycline-treated dormant mouse melanomas and human mutant BRAF melanoma cell lines treated with 2 µM vemurafenib for 20  days. We found conserved expression changes in histone methyltransferase genes ASH2, EZH2, PRMT5, SUV39H1, SUV39H2, and SYMD2 in P-ERK low, p-38 high melanoma cells following prolonged BRAF inhibition. Quantitative mass spectrometry, determined a corresponding reduction in histone Lys9 and Lys27 methylation and increase in Lys36 methylation in melanoma cell lines treated with 2 µM vemurafenib for 20  days. Thus, these changes as are part of the initiate response to BRAF inhibition and likely contribute to the survival of melanoma cells.

ASA Suppresses PGE2 in Plasma and Melanocytic Nevi of Human Subjects at Increased Risk for Melanoma.

Potential anti-inflammatory and anticarcinogenic effects of aspirin (ASA) may be suitable for melanoma chemoprevention, but defining biomarkers in relevant target tissues is prerequisite to performing randomized controlled chemoprevention trials. We conducted open-label studies with ASA in 53 human subjects with melanocytic nevi at increased risk for melanoma. In a pilot study, 12 subjects received a single dose (325 mg) of ASA; metabolites salicylate, salicylurate, and gentisic acid were detected in plasma after 4-8 h, and prostaglandin E2 (PGE2) was suppressed in both plasma and nevi for up to 24 h. Subsequently, 41 subjects received either 325 or 81 mg ASA (nonrandomized) daily for one week. ASA metabolites were consistently detected in plasma and nevi, and PGE2 levels were significantly reduced in both plasma and nevi. Subchronic ASA dosing did not affect 5" adenosine monophosphate-activated protein kinase (AMPK) activation in nevi or leukocyte subsets in peripheral blood, although metabolomic and cytokine profiling of plasma revealed significant decreases in various (non-ASA-derived) metabolites and inflammatory cytokines. In summary, short courses of daily ASA reduce plasma and nevus PGE2 and some metabolites and cytokines in plasma of human subjects at increased risk for melanoma. PGE2 may be a useful biomarker in blood and nevi for prospective melanoma chemoprevention studies with ASA.

AKT1E17K Activates Focal Adhesion Kinase and Promotes Melanoma Brain Metastasis.

Alterations in the PI3K/AKT pathway occur in up to 70% of melanomas and are associated with disease progression. The three AKT paralogs are highly conserved but data suggest they have distinct functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated E17K, E40K, and Q79K mutations in AKT1, AKT2, and AKT3 and show that mice harboring tumors expressing AKT1E17K had the highest incidence of brain metastasis and lowest mean survival. Tumors expressing AKT1E17K displayed elevated levels of focal adhesion factors and enhanced phosphorylation of focal adhesion kinase (FAK). AKT1E17K expression in melanoma cells increased invasion and this was reduced by pharmacologic inhibition of either AKT or FAK. These data suggest that the different AKT paralogs have distinct roles in melanoma brain metastasis and that AKT and FAK may be promising therapeutic targets. IMPLICATIONS: This study suggests that AKT1E17K promotes melanoma brain metastasis through activation of FAK and provides a rationale for the therapeutic targeting of AKT and/or FAK to reduce melanoma metastasis.

Aspirin Suppresses PGE2 and Activates AMP Kinase to Inhibit Melanoma Cell Motility, Pigmentation, and Selective Tumor Growth In Vivo.

There are conflicting epidemiologic data on whether chronic aspirin (ASA) use may reduce melanoma risk in humans. Potential anticancer effects of ASA may be mediated by its ability to suppress prostaglandin E2 (PGE2) production and activate 5'-adenosine monophosphate-activated protein kinase (AMPK). We investigated the inhibitory effects of ASA in a panel of melanoma and transformed melanocyte cell lines, and on tumor growth in a preclinical model. ASA and the COX-2 inhibitor celecoxib did not affect melanoma cell viability, but significantly reduced colony formation, cell motility, and pigmentation (melanin production) in vitro at concentrations of 1 mmol/L and 20 µmol/L, respectively. ASA-mediated inhibition of cell migration and pigmentation was rescued by exogenous PGE2 or Compound C, which inhibits AMPK activation. Levels of tyrosinase, MITF, and p-ERK were unaffected by ASA exposure. Following a single oral dose of 0.4 mg ASA to NOD/SCID mice, salicylate was detected in plasma and skin at 4 hours and PGE2 levels were reduced up to 24 hours. Some human melanoma tumors xenografted into NOD/SCID mice were sensitive to chronic daily ASA administration, exhibiting reduced growth and proliferation. ASA-treated mice bearing sensitive and resistant tumors exhibited both decreased PGE2 in plasma and tumors and increased phosphorylated AMPK in tumors. We conclude that ASA inhibits colony formation, cell motility, and pigmentation through suppression of PGE2 and activation of AMPK and reduces growth of some melanoma tumors in vivo This preclinical model could be used for further tumor and biomarker studies to support future melanoma chemoprevention trials in humans. Cancer Prev Res; 11(10); 629-42. ©2018 AACR.

Mutant IDH1 Promotes Glioma Formation In Vivo.

Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated gene in grade II-III glioma and secondary glioblastoma (GBM). A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R)-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled proneural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease.

Resistance mechanisms to genetic suppression of mutant NRAS in melanoma.

Targeted therapies have revolutionized cancer care, but the development of resistance remains a challenge in the clinic. To identify rational targets for combination strategies, we used an established melanoma mouse model and selected for resistant tumors following genetic suppression of NRAS expression. Complete tumor regression was observed in all mice, but 40% of tumors recurred. Analysis of resistant tumors showed that the most common mechanism of resistance was overexpression and activation of receptor tyrosine kinases (RTKs). Interestingly, the most commonly overexpressed RTK was Met and inhibition of Met overcame NRAS resistance in this context. Analysis of NRAS mutant human melanoma cells showed enhanced efficacy of cytotoxicity with combined RTK and mitogen-activated protein kinase kinase inhibition. In this study, we establish the importance of adaptive RTK signaling in the escape of NRAS mutant melanoma from inhibition of RAS and provide the rationale for combined blockade of RAS and RTK signaling in this context.

Mitogen-activated protein kinase pathway-dependent tumor-specific survival signaling in melanoma cells through inactivation of the proapoptotic protein bad.

Mitogen-activated protein kinase (MAPK) signaling regulates fundamental cellular functions including proliferation, differentiation, and survival. We have demonstrated previously that inhibiting MAPK signaling induces apoptosis in melanoma cells but not in normal melanocytes, suggesting that the MAPK pathway propagates essential survival signals in melanoma cells. Here, we report that the 90-kDa ribosomal S6 kinase (RSK), a downstream effector in the MAPK signaling cascade, phosphorylates and inactivates the Bcl-2 homology 3-only proapoptotic protein Bad, thereby mediating a MAPK-dependent tumor-specific survival signal in melanoma cells. The MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK)/RSK MAPK signaling module is constitutively hyperactivated, and Bad is maintained in its inactive state by phosphorylation at Ser(75) in a MEK/ERK/RSK-dependent manner in melanoma cells. In contrast, in normal melanocytes, Bad is highly phosphorylated at multiple residues (Ser(75), Ser(99), and Ser(118)) in a MAPK pathway-independent manner. Importantly, ectopic expression of a constitutively activated RSK mutant abrogates Bad activation and renders melanoma cells resistant to apoptosis induced by a MEK inhibitor. Furthermore, overexpressing alanine-substituted (S75A) Bad further sensitizes melanoma cells to MEK inhibitor-induced apoptosis. Our results suggest that the MAPK pathway mediates melanoma-specific survival signaling by differentially regulating RSK-mediated phosphorylation of the proapoptotic protein Bad and may present potentially selective therapeutic targets for the treatment of melanomas.

In vitro visualization and characterization of wild type and mutant IDH homo- and heterodimers using Bimolecular Fluorescence Complementation.

Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) were recently found in ~80% of WHO grade II-III gliomas and secondary glioblastomas. These mutations reduce the enzyme's ability to convert isocitrate to α-ketoglutarate and, instead, confer a novel gain-of-function resulting in the conversion of α-ketoglutarate to 2-hydroxglutarate (2-HG). However, IDH mutations exist in a heterozygous state such that a functional wild type allele is retained. Recent data suggest that the ability of mutant IDH1, but not mutant IDH2, to produce 2-HG is dependent on the activity of the retained wild type allele. In this study, we aimed to further our understanding of the interaction and function of wild type and mutant IDH heterodimers utilizing Bimolecular Fluorescence Complementation (BiFC). Dimerization of wild type and mutant IDH monomers conjugated to the N- and C-terminus of Venus protein, respectively, is directly proportional to the amount of fluorescence emitted and can be used as an approach to visualize and assess IDH dimerization. Thus, we utilized this method to visualize IDH homo- and heterodimers and to examine their cellular physiology based on subcellular localization, NADPH production, and 2-HG levels. Our results demonstrate that wild type and mutant IDH1 or IDH2 heterodimers display similar physiological characteristics to that of mutant IDH1 or IDH2 homodimers with the exception of their ability to generate NADPH. IDH1 heterodimers consistently generate NADPH whereas IDH2 heterodimers do not. However, the presence of mutant IDH1 or IDH2 in homo- or heterodimer configurations consistently generates equivalent levels of 2-HG. Our data suggest that the wild type protein is not required for the generation of 2-HG.

A phase I study of intratumoral ipilimumab and interleukin-2 in patients with advanced melanoma.

PURPOSE: Intratumoral interleukin-2 (IL-2) is effective but does not generate systemic immunity. Intravenous ipilimumab produces durable clinical response in a minority of patients, with potentially severe toxicities. Circulating anti-tumor T cells activated by ipilimumab may differ greatly from tumor-infiltrating lymphocytes activated by intratumoral ipilimumab in phenotypes and functionality. The objective of this study was to primarily assess the safety of intratumoral ipilimumab/IL-2 combination and to obtain data on clinical efficacy. RESULTS: There was no dose limiting toxicity. While local response of injected lesions was observed in 67% patients (95% CI, 40%-93%), an abscopal response was seen in 89% (95% CI, 68%-100%). The overall response rate and clinical benefit rate by immune-related response criteria (irRC) was 40% (95% CI, 10%-70%) and 50% (95% CI, 19%-81%), respectively. Enhanced systemic immune response was observed in most patients and correlated with clinical responses. EXPERIMENTAL DESIGN: Twelve patients with unresectable stages III/IV melanoma were enrolled. A standard 3+3 design was employed to assess highest tolerable intratumoral dose of ipilimumab and IL-2 based on toxicity during the first three weeks. Escalated doses of ipilimumab was injected into only one lesion weekly for eight weeks in cohorts of three patients. A fixed dose of IL-2 was injected three times a week into the same lesion for two weeks, followed by two times a week for six weeks. CONCLUSIONS: Intratumoral injection with the combination of ipilimumab/IL-2 is well tolerated and generates responses in both injected and non-injected lesions in the majority of patients.

AKT1 Activation Promotes Development of Melanoma Metastases.

Metastases are the major cause of melanoma-related mortality. Previous studies implicating aberrant AKT signaling in human melanoma metastases led us to evaluate the effect of activated AKT1 expression in non-metastatic BRAF(V600E)/Cdkn2a(Null) mouse melanomas in vivo. Expression of activated AKT1 resulted in highly metastatic melanomas with lung and brain metastases in 67% and 17% of our mice, respectively. Silencing of PTEN in BRAF(V600E)/Cdkn2a(Null) melanomas cooperated with activated AKT1, resulting in decreased tumor latency and the development of lung and brain metastases in nearly 80% of tumor-bearing mice. These data demonstrate that AKT1 activation is sufficient to elicit lung and brain metastases in this context and reveal that activation of AKT1 is distinct from PTEN silencing in metastatic melanoma progression. These findings advance our knowledge of the mechanisms driving melanoma metastasis and may provide valuable insights for clinical management of this disease.

Akt signaling accelerates tumor recurrence following ras inhibition in the context of ink4a/arf loss.

Aberrant activation of the RAS signaling pathway contributes to nearly all human cancers, including gliomas. To determine the dependence of high-grade gliomas on this signaling pathway, we developed a doxycycline-regulated KRas glioma mouse model. Using this model we previously demonstrated that inhibition of KRas expression in gliomas induced by activated KRas and Akt results in complete tumor regression. We have also shown that, in the context of Ink4a/Arf loss, abrogation of KRas signaling is sufficient to decrease tumor burden but resistance ensues. In this study, we sought to determine the effect of activated Akt signaling in combination with activated KRas and loss of Ink4a/Arf on the growth and recurrence of brain tumors following suppression of KRas expression. We observed significant tumor formation in Ink4a/Arf(lox/lox) mice injected with retroviruses containing tetracycline responsive element (TRE)-KRas, Tet-off, Akt, and Cre. Abrogation of KRas signaling resulted in significant tumor regression; however, resistance developed after a relatively short latency. Tumor recurrence occurred more rapidly and the tumors were more aggressive in the presence of activated Akt signaling compared with loss of Ink4a/Arf alone suggesting that this pathway contributes to tumor progression in this context.

Ink4a/Arf loss promotes tumor recurrence following Ras inhibition.

Aberrant activation of rat sarcoma (Ras) signaling contributes to the development of a variety of human cancers, including gliomas. To determine the dependence of high-grade gliomas on continued Ras signaling, we developed a doxycycline-regulated Kirsten Ras (KRas) glioma mouse model. We previously demonstrated that KRas is required for the maintenance of glioblastoma multiforme tumors arising in the context of activated Akt signaling in vivo; inhibition of KRas expression resulted in apoptotic tumor regression and significantly increased survival. We utilized a well-established glioma mouse model to determine the reliance of gliomas on continued KRas signaling in the context of Ink4a/Arf deficiency, a common occurrence in human gliomas. Despite the dependency of primary gliomas on continued KRas signaling, a significant percentage of tumors progressed to a KRas-independent state in the absence of Ink4a/Arf expression, demonstrating that these tumor suppressors play a critical role in the suppression of glioma recurrence. While even advanced stages of gliomas may remain dependent upon KRas signaling for maintenance and growth, our findings demonstrate that loss of Ink4a/Arf facilitates the acquisition of oncogene independence and tumor recurrence. Furthermore, reactivation of the Ras mitogen-activated protein kinase pathway in the absence of virally delivered KRas expression is a common mechanism of recurrence in this context.

Akt signaling is required for glioblastoma maintenance in vivo.

Glioblastoma multiforme (GBM) can be induced in mice through the combined expression of activated forms of KRas and Akt in glial progenitor cells. We have previously demonstrated that KRas is required for the maintenance of these tumors in vivo as inhibition of KRas expression resulted in apoptotic tumor regression and significantly increased survival. To determine the reliance of these tumors on Akt signaling in vivo, we generated a viral vector that allows the expression of Akt to be controlled post-delivery. Survival rates were compared between those animals with continued Akt expression and animals in which expression of Akt was suppressed. Although a fifth of the tumors were refractory to treatment, inhibition of Akt significantly increased the survival of tumor-bearing mice and nearly a fourth of the mice remained in remission four months after the treatment period. These data suggest that Akt is required for glioblastoma maintenance in the context of activated Ras and that loss of Akt expression results in increased survival; therefore, the PI3K/AKT signaling pathway is a viable therapeutic target in this context.

Targeted delivery of NRASQ61R and Cre-recombinase to post-natal melanocytes induces melanoma in Ink4a/Arflox/lox mice.

We have developed a somatic cell gene delivery mouse model of melanoma that allows for the rapid validation of genetic alterations identified in this disease. A major advantage of this system is the ability to model the multi-step process of carcinogenesis in immune-competent mice without the generation and cross breeding of multiple strains. We have used this model to evaluate the role of RAS isoforms in melanoma initiation in the context of conditional Ink4a/Arf loss. Mice expressing the tumor virus A (TVA) receptor specifically in melanocytes under control of the dopachrome tautomerase (DCT) promoter were crossed to Ink4a/Arf(lox/lox) mice and newborn DCT-TVA/Ink4a/Arf(lox/lox) mice were injected with retroviruses containing activated KRAS, NRAS and/or Cre-recombinase. No mice injected with viruses containing KRAS and Cre or NRAS alone developed tumors; however, more than one-third of DCT-TVA/Ink4a/Arf(lox/lox) mice injected with NRAS and Cre viruses developed melanoma and two-thirds developed melanoma when NRAS and Cre expression was linked.

Mitogen-activated protein kinase inhibition induces translocation of Bmf to promote apoptosis in melanoma.

Constitutive activation of the mitogen-activated protein kinase (MAPK) pathway is implicated in the development and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRAS or BRAF have been identified in approximately 85% of melanomas, and components of this pathway have been developed as molecular targets for therapeutic intervention. We and others have shown that inhibition of this pathway with specific small molecule MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors induces a wide range of apoptotic responsiveness in human melanoma cells both in vitro and in vivo. To define the molecular mechanism underlying variable apoptotic sensitivity of melanoma cells to MEK inhibition, we examined the expression and subcellular localization of Bcl-2 family members in a comprehensive set of human melanoma cell lines. Whereas the proapoptotic protein Bim was activated and localized to the mitochondrial membrane in all cell lines regardless of apoptotic sensitivity, Bmf activation and cytosolic translocation was exclusive to sensitive cells. In resistant cells, Bmf remained sequestered to the cytoskeleton through dynein light chain 2 (DLC2) binding. Overexpression of Bmf in resistant cells did not enhance apoptosis, whereas expression of mutant BmfA69P, which has decreased binding to DLC2, promoted cell death. Expression of BmfA69P mutants possessing the Bcl-2 homology 3 (BH3) domain mutation L138A, which impairs BH3 interactions, did not enhance apoptosis in resistant cells. RNA interference targeting Bim and Bmf provided protection from apoptosis induced by MEK inhibition. These results show a novel role for Bmf in promoting apoptosis and provide insight into the mechanism of apoptotic resistance to MEK inhibition in melanoma.