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35336 Effect of Nuclear Soluble Adenylyl Cyclase (sAC) on Melanoma Treatment Response
- Jakyung Bang, Marek M. Drozdz, Lauren Dong, Taha Merghoub, Jonathan H. Zippin
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- Journal:
- Journal of Clinical and Translational Science / Volume 5 / Issue s1 / March 2021
- Published online by Cambridge University Press:
- 30 March 2021, pp. 138-139
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ABSTRACT IMPACT: Our data identify a novel candidate for combination strategy in melanoma treatment, and can inform clinicians in their decision-making process regarding therapeutic intervention for melanoma patients. OBJECTIVES/GOALS: Soluble adenylyl cyclase (sAC) is a novel source of cyclic AMP (cAMP). In melanoma, nuclear sAC localization has an established diagnostic utility and we newly found that nuclear sAC functions as a tumor suppressor by inhibiting Hippo pathway, which affects treatment response. Here, we examine the effect of nuclear sAC on melanoma treatment response. METHODS/STUDY POPULATION: We developed a doxycycline inducible system for increasing sAC activity only in the nucleus. We assessed whether nuclear sAC activity affects treatment response, using BRAFV600 human melanoma cell lines. Using a clonogenic assay, we examined how nuclear sAC activity affects growth inhibition in the presence of a BRAF inhibitor, vemurafenib. Our findings will be confirmed in vivo using tumor xenografts. After tumor formation in NSG mice, mice will be randomized to be fed normal or doxycycline chow for nuclear sAC induction, then subdivided to receive vehicle or vemurafenib to examine the effect of nuclear sAC activity on treatment response in vivo. We will also compare melanoma biopsies collected before and after treatment with BRAF inhibitors to assess how nuclear sAC staining affects tumor morphology in vivo. RESULTS/ANTICIPATED RESULTS: So far, nuclear sAC activity has rendered SkMel178 and M263 cell lines more susceptible to vemurafenib. Cell viability was inversely correlated both with vemurafenib and with doxycycline concentration. Cell viability after vemurafenib treatment was dramatically reduced when nuclear sAC was activated. It appears that nuclear sAC enhances the sensitivity of BRAF mutant melanomas to vemurafenib in vitro. We anticipate that xenografts of these cells in mice will be more susceptible to vemurafenib when nuclear sAC is activated. We also anticipate that positive nuclear sAC staining will correlate with a favorable response to therapy. DISCUSSION/SIGNIFICANCE OF FINDINGS: Targeted therapy with BRAF inhibitors is used in late-stage melanomas, but its use is limited as patients invariably acquire resistance. Here, we identified nuclear sAC activation as a novel candidate for combination strategy. Our data will also inform clinicians how best to integrate this biomarker into their decision-making regarding therapy.
2173: Investigation of sAC signaling reveals new therapeutic targets for cancer cell metabolism
- Jenny Wang, Jonathan Zippin
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- Journal:
- Journal of Clinical and Translational Science / Volume 1 / Issue S1 / September 2017
- Published online by Cambridge University Press:
- 10 May 2018, p. 2
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OBJECTIVES/SPECIFIC AIMS: The soluble adenylyl cyclase (sAC) is a noncanonical source of cAMP in mammalian cells. sAC is an ATP/bicarbonate ion sensor, whose activity responds to intracellular signals such as pH changes and metabolism. Unlike the more traditionally studied transmembrane adenylyl cyclase, sAC is not tethered to the cell membrane and instead is found in subcellular microdomains like the mitochondria and nucleus. In particular, sAC localization in the mitochondria has been implicated in oxidative phosphorylation and mitochondrial metabolism. Specific changes in sAC microdomain localization have diagnostic utility in a wide variety of cancers, namely melanoma. We have recently found that loss of sAC leads to tumorigenesis and a Warburg/cancer-like metabolic phenotype, consisting of an activated flux through glycolysis, increased lactate production, and dependence on glucose metabolism. In addition, computational analysis of the metabolomics profile of sAC null cells suggests an increased flux through serine synthetic pathways. We hypothesized that specific sAC microdomains are responsible for this cancer-like metabolic state. METHODS/STUDY POPULATION: We have established oncogenic SV40 large T antigen and HPV16-E6 expressing mouse embryonic fibroblasts lacking sAC expression (SV40 KO and E6 KO, respectively). Using these parental lines, we reintroduced sAC by targeting the protein to specific microdomains. sAC was either driven into the mitochondria (mito-sAC) or was driven into all possible microdomains (WT sAC). Single clones were generated and sAC expression was confirmed by Western analysis. Stable cell lines were evaluated for mitochondrial metabolism, glucose sensitivity, and serine sensitivity. RESULTS/ANTICIPATED RESULTS: We found that reintroduction of WT sAC into sAC null cells rescued sensitivity to glycolytic inhibition compared with control cells (p<0.01). The effect was not dependent on the method of immortalization as it was seen in both SV40 and E6 KO cell lines. sAC activity was not directly proportional to expression suggesting that additional regulatory pathways exist. Interestingly, targeted delivery of sAC to the mitochondria was not as effective in rescuing glucose sensitivity as untargeted delivery of sAC into all possible microdomains. Therefore, even though mitochondrial sAC is known to influence metabolism, our data suggests that the nonmitochondrial isoform is most important for cancer cell metabolism. Although metabolomics analysis suggested that serine synthetic pathways are activated in sAC null cells, there is no evidence to suggest that serine is required for sAC null cell growth. Neither inhibition of serine synthesis nor serine starvation differentially affected the growth of sAC null cells compared with WT sAC. DISCUSSION/SIGNIFICANCE OF IMPACT: These data suggest that the Warburg metabolic phenotype in sAC null cells is regulated by specific sAC microdomains. By targeting sAC to specific microdomains, we can further distinguish the role of sAC localization in cellular metabolism. Cancer cells have been shown to exhibit altered metabolic circuitry of pathways like glycolysis, which allow them to adapt to increased metabolic demands of cellular proliferation and waning environmental resources. Beyond helping us improve the use of sAC immunolocalization as a cancer diagnostic, a better understanding of sAC microdomains in transformed cells will help us understand how this signaling pathway is important in cancer. Pharmacologic manipulation of sAC signaling may represent a new cancer therapeutic strategy.
2321: Soluble adenylyl cyclase (sAC) regulates melanogenesis and melanocyte response to UVB
- Koji Ota, Dalee Zhou, Jonathan Zippin
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- Journal:
- Journal of Clinical and Translational Science / Volume 1 / Issue S1 / September 2017
- Published online by Cambridge University Press:
- 10 May 2018, p. 7
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OBJECTIVES/SPECIFIC AIMS: Our objective is to study the role of soluble adenylyl cyclase in the melanocyte regulation of pigment in response to ultraviolet radiation. Melanocytes are specialized cells that produce melanin in organelles called melanosomes, and melanin determines the pigmentation of hair and skin. cAMP is a master regulator of pigmentation and transmembrane class of adenylyl cyclases are essential for expression of important enzymes involved in melanogenesis. However, pigmentation is also controlled by melanosomal pH, which regulates melanogenesis, tyrosinase activity, and melanosome maturation. The relationship between melanosomal pH and cAMP has been elusive. Soluble adenylyl cyclase is a noncanonical source of cAMP that is not responsive to G proteins but rather functions as a pH sensor. We recently demonstrated that loss of soluble adenylyl cyclase (sAC) activity leads to increased melanosomal pH as well as increased pigmentation in cells and hair. We expanded our research to investigate the role of sAC in the intrinsic response of melanocytes to ultraviolet radiation. METHODS/STUDY POPULATION: We utilized sACfl/fl (wild type) and sACKO mouse melanocytes and compared their change in pigmentation in response to ultraviolet radiation. Melanin was used as a measure of pigmentation. We irradiated these cells at differing doses of UVB (0, 1, 2, or 3 mJ/cm2) daily for 3 days. After UVB treatment, cells were observed and the surviving cell numbers were determined. Cells were then analyzed for melanin content using spectroscopy. RESULTS/ANTICIPATED RESULTS: We found that while both sACfl/fl and sACKO cells had increased melanin content in response to UVB, the melanin content of sACKO cells increased more compared with sACfl/fl cells (p=0.001 at daily dose of 3 mJ/cm2). In addition, sACKO cells required less UVB dose to induce a response. We also observed that sACKO cells show increased cell death compared with sACfl/fl cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Although both sACfl/fl and sACKO cells can induce melanin production in response to UV, our results suggest that sACKO cells are more sensitive. We believe that this increased response in sACKO cells is due to increased melanosomal pH. In addition, sACKO cells show increased cell death, suggesting that sAC is important in the damage response secondary to UV exposure. UV plays a wide range of roles in skin biology such as contributing to cancer risk and pigmentation. Since pigmentation is essential for the protection of the skin from UV insult, further investigation of possible mechanisms in which sAC can influence pigmentation in response to UV is warranted.