Marcus Bosenberg

BrafV600E cooperates with Pten loss to induce metastatic melanoma

Mutational activation of BRAF is the earliest and most common genetic alteration in human melanoma. To build a model of human melanoma, we generated mice with conditional melanocyte-specific expression of BRafV600E. Upon induction of BRafV600E expression, mice developed benign melanocytic hyperplasias that failed to progress to melanoma over 15–20 months. By contrast, expression of BRafV600E combined with Pten tumor suppressor gene silencing elicited development of melanoma with 100% penetrance, short latency and with metastases observed in lymph nodes and lungs. Melanoma was prevented by inhibitors of mTorc1 (rapamycin) or MEK1/2 (PD325901) but, upon cessation of drug administration, mice developed melanoma, indicating the presence of long-lived melanoma-initiating cells in this system. Notably, combined treatment with rapamycin and PD325901 led to shrinkage of established melanomas. These mice, engineered with a common genetic profile to human melanoma, provide a system to study melanomas cardinal feature of metastasis and for preclinical evaluation of agents designed to prevent or treat metastatic disease.

Deregulated Akt3 Activity Promotes Development of Malignant Melanoma

Malignant melanoma is the skin cancer with the most significant impact on man, carrying the highest risk of death from metastasis. Both incidence and mortality rates continue to rise each year, with no effective long-term treatment on the horizon. In part, this reflects lack of identification of critical genes involved and specific therapies targeted to correct these defects. We report that selective activation of the Akt3 protein promotes cell survival and tumor development in 43 to 60% of nonfamilial melanomas. The predominant Akt isoform active in melanomas was identified by showing that small interfering RNA (siRNA) against only Akt3, and not Akt1 or Akt2, lowered the amount of phosphorylated (active) Akt in melanoma cells. The amount of active Akt3 increased progressively during melanoma tumor progression with highest levels present in advanced-stage metastatic melanomas. Mechanisms of Akt3 deregulation occurred through a combination of overexpression of Akt3 accompanying copy number increases of the gene and decreased PTEN protein function occurring through loss or haploinsufficiency of the PTEN gene. Targeted reduction of Akt3 activity with siRNA or by expressing active PTEN protein stimulated apoptotic signaling, which reduced cell survival by increasing apoptosis rates thereby inhibiting melanoma tumor development. Identifying Akt3 as a selective target in melanoma cells provides new therapeutic opportunities for patients in the advanced stages of this disease.

Phosphoenolpyruvate Is a Metabolic Checkpoint of Anti-tumor T Cell Responses

Activated T cells engage aerobic glycolysis and anabolic metabolism for growth, proliferation, and effector functions. We propose that a glucose-poor tumor microenvironment limits aerobic glycolysis in tumor-infiltrating T cells, which suppresses tumoricidal effector functions. We discovered a new role for the glycolytic metabolite phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca(2+)-NFAT signaling and effector functions by repressing sarco/ER Ca(2+)-ATPase (SERCA) activity. Tumor-specific CD4 and CD8 T cells could be metabolically reprogrammed by increasing PEP production through overexpression of phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions. Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. This study uncovers new metabolic checkpoints for T cell activity and demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance anti-tumor T cell responses, illuminating new forms of immunotherapy.

An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background

People with pale skin, red hair, freckles and an inability to tan—the ‘red hair/fair skin’ phenotype—are at highest risk of developing melanoma, compared to all other pigmentation types. Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAFV600E, into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1re/e background. Selective absence of pheomelanin synthesis was protective against melanoma development. In addition, normal Mc1re/e mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1re/e mouse skin. These data suggest that the pheomelanin pigment pathway produces ultraviolet-radiation-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage. Although protection from ultraviolet radiation remains important, additional strategies may be required for optimal melanoma prevention.

Loss of PTEN promotes resistance to T cell–mediated immunotherapy

UNLABELLED T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell-mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti-PD-1 and anti-CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors. SIGNIFICANCE This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K-AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K-AKT pathway to increase the efficacy of immunotherapy.

Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.

Melanocyte stem cells (McSCs) intimately interact with epithelial stem cells (EpSCs) in the hair follicle bulge and secondary hair germ (sHG). Together, they undergo activation and differentiation to regenerate pigmented hair. However, the mechanisms behind this coordinated stem cell behavior have not been elucidated. Here, we identified Wnt signaling as a key pathway that couples the behavior of the two stem cells. EpSCs and McSCs coordinately activate Wnt signaling at the onset of hair follicle regeneration within the sHG. Using genetic mouse models that specifically target either EpSCs or McSCs, we show that Wnt activation in McSCs drives their differentiation into pigment-producing melanocytes, while EpSC Wnt signaling not only dictates hair follicle formation but also regulates McSC proliferation during hair regeneration. Our data define a role for Wnt signaling in the regulation of McSCs and also illustrate a mechanism for regeneration of complex organs through collaboration between heterotypic stem cell populations.

Pleomorphic Liposarcoma: Clinicopathologic Analysis of 57 Cases

Pleomorphic liposarcoma is an uncommon form of liposarcoma that only recently has been properly characterized. A series of 57 cases is presented. Patient age at presentation ranged from 27 to 95 years (median, 54 years), and there was a slight male predilection (male/female ratio = 1.2:1). Tumors most frequently involved the lower limb (47% of cases) or upper limb (18%). Other anatomic sites, including trunk (14%), retroperitoneum (7%), head and neck (5%), abdomen/pelvis (5%), and spermatic cord (4%), were less frequently involved. Tumor size ranged from 1.5 to 21 cm (median, 8 cm), with deep (subfascial) locations (39 cases) being more frequent than subcutaneous (11 cases) or dermal sites (5 cases). All lesions showed features of pleomorphic sarcoma and at least focally contained typical multivacuolated lipoblasts. Although there was considerable overlap, tumors fell into three broad categories: high-grade pleomorphic/spindle cell sarcoma with scattered lipoblasts or sheets of lipoblasts (60%), high-grade pleomorphic sarcoma with epithelioid areas and scattered lipoblasts (28%), and intermediate- to high-grade sarcoma predominantly resembling myxofibrosarcoma except for the presence of lipoblasts (12%). Immunohistochemistry revealed focal staining for smooth muscle actin in 13 of 29 cases (45%), S-100 protein positivity in lipoblasts in 15 of 45 cases (33%), focal staining for keratin in 6 of 28 cases (21%), including 5 of 13 (38%) with epithelioid morphology, and focal staining for desmin in 4 of 30 cases (13%). Follow-up data, available in 50 patients (88%) (median, 33 months), showed local recurrence in 34% of patients, systemic metastases in 32%, and tumor-related death in 32%. Only 2 of the 16 superficial (dermal or subcutaneous) lesions metastasized. Five-year overall, local recurrence-free, metastasis-free, and disease-free survivals were 63%, 58%, 58%, and 39%, respectively. By univariate analysis, central (nonextremity) location, deep situation, tumor size ≥10 cm, mitotic rate ≥10 per 10 HPF, necrosis, and epithelioid morphology were associated with a worse prognosis. However, by multivariate analysis, only age ≥60 years, central location, tumor size, and mitotic rate remained independent predictors for an adverse outcome. By multivariate analysis, wide local excision or amputation and postoperative radiotherapy protected against local recurrence.

Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas

We report on whole-exome sequencing (WES) of 213 melanomas. Our analysis established NF1, encoding a negative regulator of RAS, as the third most frequently mutated gene in melanoma, after BRAF and NRAS. Inactivating NF1 mutations were present in 46% of melanomas expressing wild-type BRAF and RAS, occurred in older patients and showed a distinct pattern of co-mutation with other RASopathy genes, particularly RASA2. Functional studies showed that NF1 suppression led to increased RAS activation in most, but not all, melanoma cases. In addition, loss of NF1 did not predict sensitivity to MEK or ERK inhibitors. The rebound pathway, as seen by the induction of phosphorylated MEK, occurred in cells both sensitive and resistant to the studied drugs. We conclude that NF1 is a key tumor suppressor lost in melanomas, and that concurrent RASopathy gene mutations may enhance its role in melanomagenesis.

HIN-1, a putative cytokine highly expressed in normal but not cancerous mammary epithelial cells

To identify molecular alterations implicated in the initiating steps of breast tumorogenesis, we compared the gene expression profiles of normal and ductal carcinoma in situ (DCIS) mammary epithelial cells by using serial analysis of gene expression (SAGE). Through the pair-wise comparison of normal and DCIS SAGE libraries, we identified several differentially expressed genes. Here, we report the characterization of one of these genes, HIN-1 (high in normal-1). HIN-1 expression is significantly down regulated in 94% of human breast carcinomas and in 95% of preinvasive lesions, such as ductal and lobular carcinoma in situ. This decrease in HIN-1 expression is accompanied by hypermethylation of its promoter in the majority of breast cancer cell lines (>90%) and primary tumors (74%). HIN-1 is a putative cytokine with no significant homology to known proteins. Reintroduction of HIN-1 into breast cancer cells inhibits cell growth. These results indicate that HIN-1 is a candidate tumor suppressor gene that is inactivated at high frequency in the earliest stages of breast tumorogenesis.

Expansion and Activation of CD103+ Dendritic Cell Progenitors at the Tumor Site Enhances Tumor Responses to Therapeutic PD-L1 and BRAF Inhibition

Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen-presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103(+) dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8(+) T cells. CD103(+) DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PD-L1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the growth factor FLT3L followed by intratumoral poly I:C injections expanded and activated CD103(+) DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103(+) DCs in tumors limits checkpoint-blockade efficacy and combined FLT3L and poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade.