The role of keratinocytic RXR[alpha] in regulating melanocyte homeostasis and carcinogen induced melanomagenesis

Cutaneous melanoma remains the deadliest form of skin cancer, with a diagnosis of metastasis indicating a median survival rate of less than a year. Solar ultraviolet (UV) radiation, especially childhood sun exposure, is an important etiological risk factor of melanoma. Previous studies determined that mice selectively lacking the nuclear hormone receptor Retinoid X Receptor α in epidermal keratinocytes (RXRα[superscript ep-/-]) developed a higher number of aggressive melanocytic tumors compared to wild type mice after two-step chemical carcinogenesis, suggesting a novel role of keratinocytic nuclear receptor signalling during melanoma progression. We then discovered a progressive loss of RXRα expression in epidermal keratinocytes during melanoma progression in humans. We also investigated the contributions of CDK4[superscript R24C/R24C] and keratinocytic RXRα to influence metastatic progression in a mouse model by generating RXRα[superscript ep-/-]/CDK4[superscript R24C/R24C] bigenic mice containing an activated cyclin dependent kinase 4 (CDK4), besides lacking RXRα in epidermal keratinocytes. Those bigenic mice developed malignant melanomas that metastasized to regional lymph nodes after carcinogen exposure. Expression of several keratinocyte-derived growth factors implicated in melanomagenesis were upregulated in the skin of bigenic mice, and recruitment of RXRα was shown on the promoters of endothelin-1 (Edn1) and hepatocyte growth factor (Hgf). We then confirmed a downregulation of factors (FAS, E-cadherin and PTEN) implicated in apoptosis, invasion and survival within the melanocytic tumors. To further evaluate the paracrine role that EDN1 has on melanocyte activation, we utilized a transgenic mouse model where the gene encoding Edn1 was selectively ablated from epidermal keratinocytes using the Cre-LoxP strategy to create the EDN1[superscript ep-/-] knockout mouse line. We discovered a direct in vivo transcriptional regulation of keratinocytic Edn1 by the tumor-suppressor p53 in epidermal keratinocytes in response to UV irradiation. We also demonstrate that in vivo disruption of keratinocyte-derived EDN1 signaling alters melanocyte proliferation and decreases epidermal and dermal melanocyte populations in both normal and UV exposed mouse skin. EDN1 also has a protective role against UVR-induced DNA damage and apoptosis and similar effects on UV-induced melanocyte proliferation and DNA damage are observed in p53-null mice. Inhibition of EDN1 signaling by topical application of an EDNRB antagonist BQ788 on mouse skin also recapitulates epidermal EDN1 ablation. Furthermore, treatment of primary murine melanocytes with BQ788 abrogates signaling downstream of this receptor. Taken together, these studies demonstrate the contribution of RXRα regulated keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes. Also, they establish an essential role of EDN1 in epidermal keratinocytes to mediate UV-induced melanocyte homeostasis in vivo

In silico and in vitro drug screening identifies new therapeutic approaches for Ewing sarcoma.

The long-term overall survival of Ewing sarcoma (EWS) patients remains poor; less than 30% of patients with metastatic or recurrent disease survive despite aggressive combinations of chemotherapy, radiation and surgery. To identify new therapeutic options, we employed a multi-pronged approach using in silico predictions of drug activity via an integrated bioinformatics approach in parallel with an in vitro screen of FDA-approved drugs. Twenty-seven drugs and forty-six drugs were identified, respectively, to have anti-proliferative effects for EWS, including several classes of drugs in both screening approaches. Among these drugs, 30 were extensively validated as mono-therapeutic agents and 9 in 14 various combinations in vitro. Two drugs, auranofin, a thioredoxin reductase inhibitor, and ganetespib, an HSP90 inhibitor, were predicted to have anti-cancer activities in silico and were confirmed active across a panel of genetically diverse EWS cells. When given in combination, the survival rate in vivo was superior compared to auranofin or ganetespib alone. Importantly, extensive formulations, dose tolerance, and pharmacokinetics studies demonstrated that auranofin requires alternative delivery routes to achieve therapeutically effective levels of the gold compound. These combined screening approaches provide a rapid means to identify new treatment options for patients with a rare and often-fatal disease

Retinoid-X-Receptors (α/β) in Melanocytes Modulate Innate Immune Responses and Differentially Regulate Cell Survival following UV Irradiation

Understanding the molecular mechanisms of ultraviolet (UV) induced melanoma formation is becoming crucial with more reported cases each year. Expression of type II nuclear receptor Retinoid-X-Receptor α (RXRα) is lost during melanoma progression in humans. Here, we observed that in mice with melanocyte-specific ablation of RXRα and RXRβ, melanocytes attract fewer IFN-γ secreting immune cells than in wild-type mice following acute UVR exposure, via altered expression of several chemoattractive and chemorepulsive chemokines/cytokines. Reduced IFN-γ in the microenvironment alters UVR-induced apoptosis, and due to this, the survival of surrounding dermal fibroblasts is significantly decreased in mice lacking RXRα/β. Interestingly, post-UVR survival of the melanocytes themselves is enhanced in the absence of RXRα/β. Loss of RXRs α/β specifically in the melanocytes results in an endogenous shift in homeostasis of pro- and anti-apoptotic genes in these cells and enhances their survival compared to the wild type melanocytes. Therefore, RXRs modulate post-UVR survival of dermal fibroblasts in a “non-cell autonomous” manner, underscoring their role in immune surveillance, while independently mediating post-UVR melanocyte survival in a “cell autonomous” manner. Our results emphasize a novel immunomodulatory role of melanocytes in controlling survival of neighboring cell types besides controlling their own, and identifies RXRs as potential targets for therapy against UV induced melanoma

Case report: Invasive fungal infection in a patient with a rare CVID-causing gene (TNFRSF13B) mutation undergoing AML treatment

Acute myeloid leukemia (AML) is a complex diagnosis that puts patients at a higher risk for developing infections, particularly invasive fungal infections (IFI). Mutations in TNFRSF13B have been shown to cause dysfunction in B-cell homeostasis and differentiation, making it a risk factor for developing immunodeficiency syndromes. In this case, a male patient in his 40s presented to our emergency department (ED) with symptoms leading to a diagnosis of AML with concurrent mucormycosis of the lungs and sinuses. Targeted next generation sequencing (NGS) of the patient’s bone marrow showed, among other variants, a loss of function mutation in the TNFRSF13B gene. While most patients present with fungal infections after prolonged periods of neutropenia associated with AML treatment, this case presented with IFI at diagnosis without neutropenia suggesting an immunodeficiency syndrome. The concurrent IFI and AML diagnoses create a delicate balance between treatment of the infection and the malignancy. This case highlights the risk of infection in patients receiving chemotherapy, especially those with unrecognized immunodeficiency syndromes, and emphasizes the importance of NGS for prognosis and treatment

Drug discovery using clinical outcome-based Connectivity Mapping: application to ovarian cancer

BackgroundEpithelial ovarian cancer (EOC) is the fifth leading cause of cancer death among women in the United States (5 % of cancer deaths). The standard treatment for patients with advanced EOC is initial debulking surgery followed by carboplatin-paclitaxel combination chemotherapy. Unfortunately, with chemotherapy most patients relapse and die resulting in a five-year overall survival around 45 %. Thus, finding novel therapeutics for treating EOC is essential. Connectivity Mapping (CMAP) has been used widely in cancer drug discovery and generally has relied on cancer cell line gene expression and drug phenotype data. Therefore, we took a CMAP approach based on tumor information and clinical endpoints from high grade serous EOC patients.MethodsWe determined tumor gene expression signatures (e.g., sets of genes) associated with time to recurrence (with and without adjustment for additional clinical covariates) among patients within TCGA (n = 407) and, separately, from the Mayo Clinic (n = 326). Each gene signature was inputted into CMAP software (Broad Institute) to determine a set of drugs for which our signature "matches" the "reference" signature, and drugs that overlapped between the CMAP analyses and the two studies were carried forward for validation studies involving drug screens on a set of 10 EOC cell lines.ResultsOf the 11 drugs carried forward, five (mitoxantrone, podophyllotoxin, wortmannin, doxorubicin, and 17-AAG) were known a priori to be cytotoxics and were indeed shown to effect EOC cell viability.ConclusionsFuture research is needed to investigate the use of these CMAP and similar analyses for determining combination therapies that might work synergistically to kill cancer cells and to apply this in silico bioinformatics approach using clinical outcomes to other cancer drug screening studies

Evolving impact of long-term survival results on metastatic melanoma treatment

Melanoma treatment has been revolutionized over the past decade. Long-term results with immuno-oncology (I-O) agents and targeted therapies are providing evidence of durable survival for a substantial number of patients. These results have prompted consideration of how best to define long-term benefit and cure. Now more than ever, oncologists should be aware of the long-term outcomes demonstrated with these newer agents and their relevance to treatment decision-making. As the first tumor type for which I-O agents were approved, melanoma has served as a model for other diseases. Accordingly, discussions regarding the value and impact of long-term survival data in patients with melanoma may be relevant in the future to other tumor types. Current findings indicate that, depending on the treatment, over 50% of patients with melanoma may gain durable survival benefit. The best survival outcomes are generally observed in patients with favorable prognostic factors, particularly normal baseline lactate dehydrogenase and/or a low volume of disease. Survival curves from melanoma clinical studies show a plateau at 3 to 4 years, suggesting that patients who are alive at the 3-year landmark (especially in cases in which treatment had been stopped) will likely experience prolonged cancer remission. Quality-of-life and mixture-cure modeling data, as well as metrics such as treatment-free survival, are helping to define the value of this long-term survival. In this review, we describe the current treatment landscape for melanoma and discuss the long-term survival data with immunotherapies and targeted therapies, discussing how to best evaluate the value of long-term survival. We propose that some patients might be considered functionally cured if they have responded to treatment and remained treatment-free for at least 2 years without disease progression. Finally, we consider that, while there have been major advances in the treatment of melanoma in the past decade, there remains a need to improve outcomes for the patients with melanoma who do not experience durable survival

Adherent cell depletion promotes the expansion of renal cell carcinoma infiltrating T cells with optimal characteristics for adoptive transfer

Background Tumor-infiltrating lymphocyte (TIL) therapy is a personalized cancer treatment which involves generating ex vivo cultures of tumor-reactive T cells from surgically resected tumors and administering the expanded TILs as a therapeutic infusion. Phase 1 of many TIL production protocols use aldesleukin (IL-2) alone to establish TIL cultures (termed “PreREP” (Pre-Rapid Expansion Protocol)); however, this fails to consistently produce TIL cultures from renal cell carcinoma (RCC) in a timely manner. Adding mitogenic stimulation via anti-CD3/anti-CD28 beads along with IL-2 to the fresh tumor digest (FTD) during TIL generation (termed “FTD+ beads”) increases successful TIL culture rates; however, T cells produced by this method may be suboptimal for adoptive transfer. We hypothesize that adherent cell depletion (ACD) before TIL expansion will produce a superior TIL product by removing the immunosuppressive signals originating from adherent tumor and stromal cells. Here we investigate if “panning,” a technique for ACD prior to TIL expansion, will impact the phenotype, functionality and/or clonality of ex vivo expanded RCC TILs.Methods Tumor specimens from 55 patients who underwent radical or partial nephrectomy at the University of Kansas Medical Center (KUMC) were used to develop the panning method and an additional 19 specimens were used to validate the protocol. Next-generation sequencing, immunohistochemistry/immunocytochemistry and flow cytometry were used during method development. The phenotype, functionality and clonality of autologous TILs generated in parallel by panning, PreREP, and FTD+ beads were assessed by flow cytometry, in vitro co-culture assays, and TCRB CDR3 sequencing.Results TIL cultures were successfully generated using the panning protocol from 15/16 clear cell, 0/1 chromophobe, and 0/2 papillary RCC samples. Significantly fewer regulatory (CD4+/CD25+/FOXP3+) (p=0.049, p=0.005), tissue-resident memory (CD8+/CD103+) (p=0.027, p=0.009), PD-1+/TIM-3+ double-positive (p=0.009, p=0.011) and TIGIT+ T cells (p=0.049, p=0.026) are generated by panning relative to PreREP and FTD+ beads respectively. Critically, a subset of TILs generated by panning were able to degranulate and/or produce interferon gamma in response to autologous tumor cells and the average tumor-reactive TIL yield was greatest when using the panning protocol.Conclusions Removing immunosuppressive adherent cells within an RCC digest prior to TIL expansion allow for the rapid production of tumor-reactive T cells with optimal characteristics for adoptive transfer

Ethical Appraisal of Credit-Card Marketing Strategy:Measure and Antecedent Exploration of Consumer Vulnerability to Credit-Card Debt

The dose response data for cotinine across the 10 EOC cell lines. (PNG 1793 kb

ColemanDanielPharmacyRetinoid-X-Receptors.pdf

Understanding the molecular mechanisms of ultraviolet (UV) induced melanoma formation is becoming crucial with more reported cases each year. Expression of type II nuclear receptor Retinoid-X-Receptor α (RXRα) is lost during melanoma progression in humans. Here, we observed that in mice with melanocyte-specific ablation of RXRα and RXRβ, melanocytes attract fewer IFN-γ secreting immune cells than in wild-type mice following acute UVR exposure, via altered expression of several chemoattractive and chemorepulsive chemokines/cytokines. Reduced IFN-γ in the microenvironment alters UVR-induced apoptosis, and due to this, the survival of surrounding dermal fibroblasts is significantly decreased in mice lacking RXRα/β. Interestingly, post-UVR survival of the melanocytes themselves is enhanced in the absence of RXRα/β. Loss of RXRs α/β specifically in the melanocytes results in an endogenous shift in homeostasis of pro- and anti-apoptotic genes in these cells and enhances their survival compared to the wild type melanocytes. Therefore, RXRs modulate post-UVR survival of dermal fibroblasts in a ‘‘non-cell autonomous’’ manner, underscoring their role in immune surveillance, while independently mediating post-UVR melanocyte survival in a ‘‘cell autonomous’’ manner. Our results emphasize a novel immunomodulatory role of melanocytes in controlling survival of neighboring cell types besides controlling their own, and identifies RXRs as potential targets for therapy against UV induced melanoma