Browsing by Subject "AMPK"

Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.

We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.

In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.

The incidence of obesity and associated diseases such as type 2-diabetes and hypertension has reached epidemic portions worldwide and attracted increased interest to understand the mechanisms that are responsible for these diseases. Obesity can result from excessive energy intake, and increasing evidence has emphasized the role of the central nervous system, especially the hypothalamus, in regulating food intake. White adipose, as a direct target of obesity and an important endocrine organ, also has long been a subject of scientific inquiry. AMPK, a conserved energy sensor, has been shown to play important roles in both the hypothalamus and adipose. Recently, CaMKK2 was shown to function as an AMPK kinase. I used intracerebroventricular cannulation as a means to acutely inhibit hypothalamic CaMKK2 with STO-609 and characterize the appetite change associated with loss of CaMKK2 function. Infusion of STO-609 in wild-type mice, but not CaMKK2-null mice, inhibited appetite and promoted weight loss consistent with reduced NPY and AgRP mRNA. Furthermore, intraperitoneal injection of ghrelin increased food intake in wild-type but not CaMKK2-null mice, and 2-DG increased appetite in both types of mice, indicating that CaMKK2 functions downstream of ghrelin to activate AMPK and upregulate appetite. As CaMKK2-null mice were protected from high-fat diet-induced obesity and diabetes, I performed a pair feeding experiment using a high-fat diet and demonstrated that protection of CaMKK2-null mice did not require reduced food consumption. Analysis of brown adipose tissue and metabolic analysis indicated that CaMKK2-null mice did not expend more energy than WT mice. Interestingly, we were surprised to find that CaMKK2-null mice had more adipose than wild-type mice when fed standard chow (5001). By real-time PCR and immunoblot, I identified CaMKK2 expression in preadipocytes and showed that it decreased during adipogenesis. I used STO-609 or shRNA to block CaMKK2 activity in preadipocytes, which resulted in enhanced adipogenesis and increased mRNA of adipogenic genes. I also identified AMPK as the relevant downstream target of CaMKK2 involved in inhibiting adipogenesis via a pathway that maintained Pref-1 mRNA. Consistent with the in vitro data, we further demonstrated that CaMKK2-null mice have more adipocytes but fewer preadipocytes, which supports our hypothesis that loss of CaMKK2 enhances adipogenesis by depleting the preadipocyte pool. Together the data presented herein contribute to our understanding of distinct mechanisms by which CaMKK2 contributes to feeding behavior and adipogenesis.

Germinal center kinases (GCKs) belong to the mammalian Ste20-like family of serine/threonine kinases and participate in various signaling pathways needed to regulate a wide range of cellular activities. GCK-like kinase (GLK), also known as MAP4K3, belongs to the MAP kinase kinase kinase kinase (MAP4K) family of proteins and has recently been established as a key node in the amino acid response pathway and putative nutrient sensing regulator in cells, as it is required for the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1)—a central regulator of cell growth and metabolism. The precise mechanism(s) by which MAP4K3 activates mTORC1 under conditions of amino acid satiety, however, are undefined. Recent studies in the La Spada lab suggest MAP4K3 activates mTORC1 by phosphorylating the NAD-dependent deacetylase sirtuin 1 (Sirt1) and subsequently, inhibiting the LKB1-AMPK pathway—a pathway that suppresses mTORC1 activation during starvation. MAP4K3 has additionally been linked to the regulation of cellular stress responses, autophagy, growth, survival, and organismal lifespan through largely unknown pathways. My working hypothesis is that MAP4K3 serves as an amino acid sensor and activates mTORC1 through phosphorylation of Sirt1 and subsequent inhibition of the mTORC1-suppressing Sirt1-LKB1-AMPK pathway under conditions of amino acid satiety and engages different biological pathways by virtue of its protein interacting partners to control critical cellular processes involved in cell growth, survival, and lifespan. In study 1, I used amino acid depletion/restimulation experiments and phospho mass spectrometry to establish a direct link between MAP4K3 and the Sirt1-LKB1-AMPK pathway and determines that Sirt1 is phosphorylated at Threonine 344 (T344) in a MAP4K3- and amino acid-dependent manner. Furthermore, I showed that phosphorylation of T344 inhibits Sirt1 and is sufficient to restore amino acid-dependent mTORC1 activation in cells lacking MAP4K3. To elucidate additional pathways regulated by MAP4K3, in study 2, I sought to discover novel MAP4K3 interacting partners by integrating proteomics interactome data and phosphoproteomics data followed by validation studies in cells. Experiments from these studies indicate a novel role for MAP4K3 in regulating DNA double-strand break (DSB) sensing and repair in the nucleus, mTOR localization to the lysosome through the GATOR2 complex, and endocytosis. Recent discoveries regarding the important role for MAP4K3 in nutrient sensing through mTORC1 activation and other cellular activities, including cell growth, autophagy, and survival are significant because deregulation of these cellular processes has been implicated in aging, as well as a wide array of human diseases including cancer, immunological disorders, and neurodegeneration. This dissertation, thus, sheds light on the molecular mechanisms by which MAP4K3 regulates these processes and provides significant insight into the modulation of these pathways in health and disease states.