Calmodulin-dependent Protein Kinase Kinase 2 Research Articles

Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CaMKK2) Expressed in the Host Promotes Lymphoma Cells Growth By Controlling Myeloid Derived Suppressor Cells Expansion

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immunosuppressive cells that generate from myeloid progenitors under pathological conditions including cancers.In response to tumor-derived factors, myeloid progenitors differentiate intoimmature myeloid cells and accumulate as monocyte and granulocyte-MDSC (M- and G-MDSC), and correlate with poor survival in patients. Promoting the differentiation of MDSCs towards mature dendritic cells and macrophage is a potential way to eliminate MDSCs and restore anti-tumor immunity. In this study, we demonstrate that the enzyme Calcium/Calmodulin dependent Kinase Kinase 2 (CaMKK2) as a potential therapeutic target to control the growth of lymphoma by regulating the expansion of MDSCs.CaMKK2 is a crucial kinase in the calcium-signaling cascade. Activated by the Ca2+/CaM complex, CaMKK2 can phosphorylate the down stream targets including CaMKI, CaMKIV, and 5' AMP-activated kinase (AMPK). Beyond central nerve system, CaMKK2 is expressed in hematopoietic stem and progenitors cells, and some hematopoietic mature cells including macrophages and monocytes. In adipogenesis, granulopoiesis, and muscle development, CaMKK2 inhibits the differentiation of progenitors towards mature cells. We reasoned that CaMKK2 might have a similar function in myeloid cells, controlling terminal differentiation of tumor-induced MDSCs.We first confirmedthe expression of CaMKK2 in tumor-induced MDSCs. EG.7-OVA lymphoma cells were injected s.c. in the flank of Camkk2-egfp reporter mice, and MDSCs subsets from the tumors and spleens of the mice were assessed by flow cytometry. We found that Camkk2 promoter was highly active in Ly6Chigh/Ly6Gdim subset (M-MDSC), but low in Ly6Cdim/Ly6Ghigh subset (G-MDSC) and Ly6Cneg/Ly6Gneg/F4/80dim/neg/MHC-IIdim/neg cells. (Figure 1) Camkk2 promoter was not active in lymphocytes as previous reported.To determine the function of CaMKK2 expressed in the host, E.G7 cells with luciferase expression were injected into the flank of wild type (WT) and Camkk2-/- mice, and monitored by tumor size and bioluminescent imaging. (Figure 2 A&B) In WT mice injected with E.G7-OVA cells, tumors were detectable by bioluminescence by day 7, and gradually increased in photon signal during the following two weeks. Although tumors successfully engrafted in Camkk2-/- mice, bioluminescent signal gradually decreased after 21 days and eventually disappeared after 32 days. (Figure 2A) In LyM-Cre+Camkk2loxP mice which Camkk2 is selectively deleted in myeloid cell compartment, when challenged with E.G7-OVA cells, similar E.G7 tumor grow suppression was observed as in the Camkk2-/- global knockout model. (Figure 2C)We next demonstrated that Camkk2 deletion decreased MDSCs accumulation in EG7 bearing mice. In normal conditions, Camkk2-/- mice had comparable CD11b+and Ly6ChighLy6Gdim percentage in the spleenswith WT mice, but slightly higher Ly6Cdim Ly6Ghigh percentage. A significant increase of MDSCs percentage was detected in EG.7-bearing mice compared to tumor-free mice; while in Camkk2-/- mice, the MDSC accumulation in E.G7-OVA spleens and tumors showed a significant decrease in percentage compared to WT ones. (Figure 3 A & B) To evaluate whether the failure of MDSC expansion in tumor-bearing Camkk2-/- mice regulated tumor suppression, MDSCs were isolated from the spleens of WT and Camkk2-/- E.G7-OVA-bearing mice, adoptively transferred into Camkk2-/- mice, and followed by tumor inoculation. As expected, lymphoma cells failed to growth in Camkk2-/- control mice without MDSC injection. However, tumors developed in all Camkk2-/- mice that received either WT or Camkk2-/- MDSCs, with no significant difference. (Figure 3C) These results indicated that restoring the number of MDSC was sufficient to revert the protective effect of Camkk2 deletion on tumor growth. Interestingly, this data also suggested that WT and Camkk2-/- MDSCs have a comparable immunosuppressive activity at single-cell level.In conclusion, our results identifies for the first time CaMKK2 as an important component of molecular machinery that mediates the detrimental effect of lymphoma cells on myeloid development. Of note, our data also pinpoint CaMKK2 as a new pharmacological target to limit the expansion of tumor-induced MDSC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Loss of Ca2+/Calmodulin Dependent Protein Kinase Kinase 2 Leads to Aberrant Transferrin Phosphorylation and Trafficking: A Potential Biomarker for Alzheimer's Disease.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a serine/threonine kinase that is activated following an increase in the intracellular Ca2+ concentration and activates multiple signaling cascades that control physiologically important neuronal processes. CaMKK2 has been implicated in schizophrenia, bipolar disease, neurodegeneration, and cancer. Using isoelectric focusing (IEF) and mass spectrometry-based proteomic analysis, it was found that knockdown (KD) of CaMKK2 in cultured adult primary dorsal root ganglion (DRG) neurons resulted in the reduction of transferrin (TF) phosphorylation at multiple functionally relevant residues which corresponded to loss of an acidic fraction (pH~3-4) of TF. In vitro studies using CRISPR/Cas9 based CaMKK2 knockout (KO) HEK293 and HepG2 cells lines validated previous findings and revealed that loss of CaMKK2 interfered with TF trafficking and turnover. TF is an iron transporter glycoprotein. Abnormal accumulation of iron and/or deregulated Ca2+ homeostasis leads to neurodegeneration in Alzheimer's disease (AD). Therefore, it was hypothesized that aberrant CaMKK2 in AD may lead to aberrant phosphorylated transferrin (P-TF: pH~3-4 fraction) which may serve as a hallmark biomarker for AD. A significant reduction of P-TF in the brain and serum of CaMKK2 KO mice and a triple-transgenic mouse model of AD (3xTg-AD) supported this hypothesis. In addition, analysis of early (< 65 years) and late-stage (>65 years) postmortem human AD cerebrospinal fluid (CSF) and serum samples revealed that aberrant P-TF (pH~3-4 fraction) profile was associated with both early and late-stage AD compared to age-matched controls. This indicates P-TF (pH~3-4 fraction) profile may be useful as a minimally invasive biomarker for AD. In addition, this study provides a link between aberrant CaMKK2 with TF trafficking and turnover which provides a novel insight into the neurodegeneration process.

Implication and Regulation of AMPK during Physiological and Pathological Myeloid Differentiation.

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase consisting of the arrangement of various α β, and γ isoforms that are expressed differently depending on the tissue or the cell lineage. AMPK is one of the major sensors of energy status in mammalian cells and as such plays essential roles in the regulation of cellular homeostasis, metabolism, cell growth, differentiation, apoptosis, and autophagy. AMPK is activated by two upstream kinases, the tumor suppressor liver kinase B1 (LKB1) and the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) through phosphorylation of the kinase on Thr172, leading to its activation. In addition, AMPK inhibits the mTOR pathway through phosphorylation and activation of tuberous sclerosis protein 2 (TSC2) and causes direct activation of unc-51-like autophagy activating kinase 1 (ULK1) via phosphorylation of Ser555, thus promoting initiation of autophagy. Although it is well established that AMPK can control the differentiation of different cell lineages, including hematopoietic stem cells (HSCs), progenitors, and mature hematopoietic cells, the role of AMPK regarding myeloid cell differentiation is less documented. The differentiation of monocytes into macrophages triggered by colony stimulating factor 1 (CSF-1), a process during which both caspase activation (independently of apoptosis induction) and AMPK-dependent stimulation of autophagy are necessary, is one noticeable example of the involvement of AMPK in the physiological differentiation of myeloid cells. The present review focuses on the role of AMPK in the regulation of the physiological and pathological differentiation of myeloid cells. The mechanisms of autophagy induction by AMPK will also be addressed, as autophagy has been shown to be important for differentiation of hematopoietic cells. In addition, myeloid malignancies (myeloid leukemia or dysplasia) are characterized by profound defects in the establishment of proper differentiation programs. Reinduction of a normal differentiation process in myeloid malignancies has thus emerged as a valuable and promising therapeutic strategy. As AMPK seems to exert a key role in the differentiation of myeloid cells, notably through induction of autophagy, we will also discuss the potential to target this pathway as a pro-differentiating and anti-leukemic strategy in myeloid malignancies.

Is TAK1 a Direct Upstream Kinase of AMPK?

Alongside Liver kinase B1 (LKB1) and Ca2+/Calmodulin-dependent protein kinase kinase 2 (CaMKK2), Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) has been suggested as a direct upstream kinase of AMP-activated protein kinase (AMPK). Several subsequent studies have reported on the TAK1-AMPK relationship, but the interpretation of the respective data has led to conflicting views. Therefore, to date the acceptance of TAK1 as a genuine AMPK kinase is lagging behind. This review provides with argumentation, whether or not TAK1 functions as a direct upstream kinase of AMPK. Several specific open questions that may have precluded the consensus are discussed based on available data. In brief, TAK1 can function as direct AMPK upstream kinase in specific contexts and in response to a subset of TAK1 activating stimuli. Further research is needed to define the intricate signals that are conditional for TAK1 to phosphorylate and activate AMPKα at T172.

Abstract 3987: CaMKK2 inhibition as a "dual-hit" strategy against ADT-induced osteoporosis and bone-metastatic prostate cancer

Abstract Prostate cancer (PCa), the most frequently diagnosed cancer in men, will have an estimated 26,730 deaths in the US during 2017. Predominantly, these deaths will be caused due to metastatic disease. PCa preferentially metastasizes to the bone undermining its structural integrity. PCa cells rely on androgens for survival and proliferation. Consequently, androgen deprivation therapy (ADT) is the first line of defense against advanced-stage PCa. However, ADT contributes to significant decreases in bone mass, predisposing the patient to an increased risk of fractures. Hence, there is a critical need for therapies that protect against bone metastatic disease as well as reverse ADT-induced bone loss in PCa patients. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a direct target of the androgen receptor (AR) and is highly over-expressed in androgen-dependent and independent forms of PCa. In vitro studies have shown that its inhibition suppresses the growth and migration of PCa cells. We observed that CaMKK2 inhibition suppresses 3-D spheroid formation by the androgen-independent PCa cell line C4-2 but not by the androgen-dependent PC3 cells. However, the exact downstream mechanism by which CaMKK2 regulates PCa growth remains unknown. More recently, CaMKK2 was shown to play a role in both the anabolic and catabolic pathways of bone remodeling. Genetic ablation and pharmacological inhibition of CaMKK2 via STO-609 stimulates osteoblasts and suppresses osteoclasts and confers protection from age- and ovariectomy-induced bone loss in mice. Based on these preliminary data, we hypothesize that inhibition of a single target, CaMKK2, will result in the therapeutic alleviation of two chief complications in advanced-stage PCa, i.e., bone metastatic tumor burden and ADT-induced bone loss. We performed sham or bilateral orchiectomy (ORX) on pretreated (saline/STO-609) 5-week-old male athymic mice (n=15 per cohort). Tri-weekly intraperitoneal (i.p.) injections were continued for 12 weeks. Additionally, two weeks after surgery, sham and ORX mice were intra-tibially injected with C4-2B cells. Micro-computed tomography analyses indicated a prevention of ORX-induced bone loss in STO-609 treated mice compared to saline treated controls (3-fold, p&amp;lt;0.05). Radiographic and histomorphomteric analyses reveal a decrease in C4-2B-initiated bone lesions in STO-609 treated mice compared to the saline treated cohorts. Taken together, our studies represent a highly novel and unique approach in the treatment of advanced-stage PCa. Citation Format: Ushashi Dadwal, Justin Williams, Austin Pucylowski, Eric Chang, Khalid Mohammad, Theresa Guise, Uma Sankar. CaMKK2 inhibition as a "dual-hit" strategy against ADT-induced osteoporosis and bone-metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3987.

Androgen Receptor-CaMKK2 Axis in Prostate Cancer and Bone Microenvironment.

The skeletal system is of paramount importance in advanced stage prostate cancer (PCa) as it is the preferred site of metastasis. Complex mechanisms are employed sequentially by PCa cells to home to and colonize the bone. Bone-resident PCa cells then recruit osteoblasts (OBs), osteoclasts (OCs), and macrophages within the niche into entities that promote cancer cell growth and survival. Since PCa is heavily reliant on androgens for growth and survival, androgen-deprivation therapy (ADT) is the standard of care for advanced disease. Although it significantly improves survival rates, ADT detrimentally affects bone health and significantly increases the risk of fractures. Moreover, whereas the majority patients with advanced PCa respond favorably to androgen deprivation, most experience a relapse of the disease to a hormone-refractory form within 1–2 years of ADT. The tumor adapts to surviving under low testosterone conditions by selecting for mutations in the androgen receptor (AR) that constitutively activate it. Thus, AR signaling remains active in PCa cells and aids in its survival under low levels of circulating androgens and additionally allows the cancer cells to manipulate the bone microenvironment to fuel its growth. Hence, AR and its downstream effectors are attractive targets for therapeutic interventions against PCa. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2), was recently identified as a key downstream target of AR in coordinating PCa cell growth, survival, and migration. Additionally, this multifunctional serine/threonine protein kinase is a critical mediator of bone remodeling and macrophage function, thus emerging as an attractive therapeutic target downstream of AR in controlling metastatic PCa and preventing ADT-induced bone loss. Here, we discuss the role played by AR-CaMKK2 signaling axis in PCa survival, metabolism, cell growth, and migration as well as the cell-intrinsic roles of CaMKK2 in OBs, OCs, and macrophages within the bone microenvironment.

Etoposide-induced protein 2.4 functions as a regulator of the calcium ATPase and protects pancreatic β-cell survival

Calcium homeostasis is essential for maintaining the viability and function of pancreatic β cells and plays a key role in preventing the development of diabetes. Decreased levels of ATPase sarcoplasmic/endoplasmic reticulum Ca2+-transporting 2 (ATP2a2), the main calcium pump in β cells, are often found in individuals with diabetes and in diabetic animal models. However, the regulators of ATP2a2 and the molecular mechanisms responsible for controlling ATP2a2 activity remain unclear. Etoposide-induced protein 2.4 (Ei24) is also down-regulated in β cells of diabetic individuals, whereas the effect of decreased Ei24 level on β-cell function is not clarified. Here, using Cre-LoxP and CRISPR/Cas9-based genomic knockout (KO) approaches to generate pancreatic β cell-specific Ei24 KO mice and pancreatic β-cell lines, we found that Ei24 regulates ATP2a2 activity. Specifically, we observed that Ei24 binds to ATP2a2 through Ei24 residues 293-299, which we named here the ATP2a2-interacting region (AIR). Loss of Ei24 inactivated ATP2a2, disrupted calcium homeostasis, and deactivated the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-AMP-activated protein kinase (AMPK) pathway. Elevation of calcium concentration in the endoplasmic reticulum or agonist-induced AMPK activation rescued pancreatic β-cell survival and improved glucose tolerance of Ei24 KO mice. Our findings indicate that targeting the Ei24-ATP2a2 interaction to increase ATP2a2 activity can protect pancreatic β cells and improve glucose homeostasis in diabetic models, suggesting that Ei24 could potentially serve as a target to prevent or manage diabetes.

1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors.

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.

Calmodulin-dependent protein kinase kinase 2 as a novel therapeutic target against osteoarthritis

Purpose: Osteoarthritis (OA) is a disabling disease that affects more than 250 million people worldwide causing tremendous individual and socioeconomic burden. OA is a complex disease encompassing multiple molecular/biological mechanisms that result in progressive cartilage loss, subchondral bone remodeling, joint tissue inflammation, and osteophytes formation. OA is often diagnosed at a late stage and treatment is confined to management of symptoms due to a lack of clinically available therapies to stop or delay the disease progression. Thus, there is a critical need to identify novel therapeutic targets that could prevent and/or delay multiple facets of OA pathogenesis. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) plays an important role in bone remodeling and macrophage-mediated inflammation. Hence, we hypothesized that genetic ablation or pharmacological inhibition of CaMKK2 will suppress inflammation and subchondral bone remodeling associated with OA. Methods: Male wild-type (WT) and/or Camkk2-/- mice (both C57BL6; n = 5 per group) underwent unilateral destabilization of medical meniscus (DMM) or sham surgery. WT mice undergoing sham and DMM surgeries were administered STO-609 (CaMKK2 inhibitor; 10 μM/kg body weight) or saline solution tri-weekly by i.p injection for 8 weeks. Damage to the joint tissues was evaluated using microcomputed tomography (microCT) and histology. The impact of CaMKK2 deficiency in cartilage pathophysiology was assessed in vitro using primary WT and Camkk2-/- murine articular chondrocytes (MACs) stimulated by interleukin-1β (IL-1β; 10 ng/ml). Quantitative real-time PCR was used to assess the expression of markers of cartilage matrix and turnover as well as inflammation. Results: Three dimensional microCT analysis of tibial subchondral bone showed a significant increase of tissue volume (TV; by 40%) and bone volume (BV/TV; 18%) in saline-treated DMM mice relative to sham-operated control mice. In addition, the thickness of subchondral bone plate (SPB) was 32% higher in DMM mice compared to sham operated cohorts. These results demonstrated an enhanced subchondral bone sclerosis after DMM surgery. Interestingly, such increases in TV and BV/TV were significantly diminished in CaMKK2 inhibitor-treated WT and Camkk2-/- mice, indicating protection from subchondral bone sclerosis induced by DMM. Moreover, SBP thickness was significantly lower in STO-609 treated WT and Camkk2-/- mice, compared to saline treated WT cohorts. Histological analyses revealed lower OARSI score and prevention of cartilage breakdown following DMM surgery in STO-609 treated WT and Camkk2-/- mice. Moreover, inhibition or loss of CaMKK2 also resulted in significantly reduced macrophage infiltration and inflammation of the synovium following DMM. Further, data obtained from isolated MACs reveal that the genetic ablation of CaMKK2 was able to counteract the effects of IL-1β by affecting the expression levels of inflammatory (iNOS, COX2, TNF-α, and IL6) cytokines and markers of cartilage catabolism (MMP2, MMP-13, and ADAMTS5). In addition, Camkk2-/- MACs expressed significantly higher levels of extracellular matrix proteins such as Col2a1, COMP and glycosaminoglycans (GAGs) in response to IL-1β, compared to WT. Whereas an increase in the activation of ERK was observed, the activity of p38 MAPK was significantly inhibited in theCamkk2-/- MACs. Inhibition of ERK with a specific inhibitor resulted in an increase of inflammatory and catabolism markers in Camkk2-/- MACs, indicating that the protective effects of CaMKK2 loss on OA pathogenesis is potentially through its regulation of MAPK activity. Conclusions: In conclusion, targeting CaMKK2 offers an overall protection of joint tissues in trauma-induced OA, owing to a suppression of inflammation and catabolism through a regulation of the ERK pathway as well as an increase in cartilage matrix production. Our study provides novel information on the role of CaMKK2 in OA pathogenesis and opens new avenues to design innovative therapeutic strategies against OA.

Chemerin suppresses neuroinflammation and improves neurological recovery via CaMKK2/AMPK/Nrf2 pathway after germinal matrix hemorrhage in neonatal rats

Chemerin, an adipokine, has been reported to reduce the production of pro-inflammatory cytokines and neutrophil infiltration. This study investigated the role of Chemerin and its natural receptor, ChemR23, as well as its downstream mediator calmodulin-dependent protein kinase kinase 2 (CAMKK2)/adenosine monophosphate-activated protein kinase (AMPK) /Nuclear factor erythroid 2-related factor 2 (Nrf2) following germinal matrix hemorrhage (GMH) in neonatal rats, with a specific focus on inflammation. GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U) in P7 rat pups. The results demonstrated that human recombinant Chemerin (rh-Chemerin) improved neurological and morphological outcomes after GMH. Rh-Chemerin promoted accumulation and proliferation of M2 microglia in periventricular regions at 72 h. Rh-Chemerin increased phosphorylation of CAMKK2, AMPK and expression of Nrf2, and decreased IL-1beta, IL-6 and TNF-alpha levels. Selective inhibition of ChemR23/CAMKK2/AMPK signaling in microglia via intracerebroventricular delivery of liposome-encapsulated specific ChemR23 (Lipo-alpha-NETA), CAMKK2 (Lipo-STO-609) and AMPK (Lipo-Dorsomorphin) inhibitor increased the expression levels of IL-1beta, IL-6 and TNF- alpha, demonstrating that ChemR23/CAMKK2/AMPK signaling in microglia suppressed inflammatory response after GMH. Cumulatively, these data showed that rh-Chemerin ameliorated GMH-induced inflammatory response by promoting ChemR23/CAMKK2/AMPK/Nrf2 pathway, and M2 microglia may be a major mediator of this effect. Thus, rh-Chemerin can serve as a potential agent to reduce the inflammatory response following GMH.

GLUT12 promotes prostate cancer cell growth and is regulated by androgens and CaMKK2 signaling.

Despite altered metabolism being an accepted hallmark of cancer, it is still not completely understood which signaling pathways regulate these processes. Given the central role of androgen receptor (AR) signaling in prostate cancer, we hypothesized that AR could promote prostate cancer cell growth in part through increasing glucose uptake via the expression of distinct glucose transporters. Here, we determined that AR directly increased the expression of SLC2A12, the gene that encodes the glucose transporter GLUT12. In support of these findings, gene signatures of AR activity correlated with SLC2A12 expression in multiple clinical cohorts. Functionally, GLUT12 was required for maximal androgen-mediated glucose uptake and cell growth in LNCaP and VCaP cells. Knockdown of GLUT12 also decreased the growth of C4-2, 22Rv1 and AR-negative PC-3 cells. This latter observation corresponded with a significant reduction in glucose uptake, indicating that additional signaling mechanisms could augment GLUT12 function in an AR-independent manner. Interestingly, GLUT12 trafficking to the plasma membrane was modulated by calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-5'-AMP-activated protein kinase (AMPK) signaling, a pathway we previously demonstrated to be a downstream effector of AR. Inhibition of CaMKK2-AMPK signaling decreased GLUT12 translocation to the plasma membrane by inhibiting the phosphorylation of TBC1D4, a known regulator of glucose transport. Further, AR increased TBC1D4 expression. Correspondingly, expression of TBC1D4 correlated with AR activity in prostate cancer patient samples. Taken together, these data demonstrate that prostate cancer cells can increase the functional levels of GLUT12 through multiple mechanisms to promote glucose uptake and subsequent cell growth.

Inhibition of CaMKK2 Enhances Fracture Healing by Stimulating Indian Hedgehog Signaling and Accelerating Endochondral Ossification.

Approximately 10% of all bone fractures do not heal, resulting in patient morbidity and healthcare costs. However, no pharmacological treatments are currently available to promote efficient bone healing. Inhibition of Ca2+ /calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) reverses age-associated loss of trabecular and cortical bone volume and strength in mice. In the current study, we investigated the role of CaMKK2 in bone fracture healing and show that its pharmacological inhibition using STO-609 accelerates early cellular and molecular events associated with endochondral ossification, resulting in a more rapid and efficient healing of the fracture. Within 7 days postfracture, treatment with STO-609 resulted in enhanced Indian hedgehog signaling, paired-related homeobox (PRX1)-positive mesenchymal stem cell (MSC) recruitment, and chondrocyte differentiation and hypertrophy, along with elevated expression of osterix, vascular endothelial growth factor, and type 1 collagen at the fracture callus. Early deposition of primary bone by osteoblasts resulted in STO-609-treated mice possessing significantly higher callus bone volume by 14 days following fracture. Subsequent rapid maturation of the bone matrix bestowed fractured bones in STO-609-treated animals with significantly higher torsional strength and stiffness by 28 days postinjury, indicating accelerated healing of the fracture. Previous studies indicate that fixed and closed femoral fractures in the mice take 35 days to fully heal without treatment. Therefore, our data suggest that STO-609 potentiates a 20% acceleration of the bone healing process. Moreover, inhibiting CaMKK2 also imparted higher mechanical strength and stiffness at the contralateral cortical bone within 4 weeks of treatment. Taken together, the data presented here underscore the therapeutic potential of targeting CaMKK2 to promote efficacious and rapid healing of bone fractures and as a mechanism to strengthen normal bones. © 2018 American Society for Bone and Mineral Research.

Sipjeondaebo-tang Alleviates Oxidative Stress-Mediated Liver Injury through Activation of the CaMKK2-AMPK Signaling Pathway.

Sipjeondaebo-tang (SDT) is used frequently as a herbal prescription to treat deficiency syndromes in traditional Korean medicine. We investigated the hepatoprotective effects of SDT against oxidative stress and attempted to clarify the underlying molecular mechanisms. SDT pretreatment reduced arachidonic acid (AA) plus iron-mediated cytotoxicity in a concentration-dependent manner and prevented changes in apoptosis-related protein expression. In addition, SDT pretreatment significantly reduced glutathione depletion, hydrogen peroxide production, and mitochondrial dysfunction via treatment with AA plus iron. SDT increased the phosphorylation of AMP-activated protein kinase (AMPK) in accordance with the phosphorylation of Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Experiments using an AMPK chemical inhibitor (Compound C) or CaMKK2 chemical inhibitor (STO-609) suggested that the CaMKK2-AMPK signaling pathway contributes to SDT-mediated protection of mitochondria and cells. Moreover, administration of SDT for 4 consecutive days to mice significantly reduced the alanine aminotransferase and aspartate aminotransferase activities induced by carbon tetrachloride, and the numbers of degenerated hepatocytes, infiltrated inflammatory cells, nitrotyrosine-positive cells, and 4-hydroxynonenal-positive cells in liver tissue. Therefore, SDT protects hepatocytes from oxidative stress via CaMKK2-dependent AMPK activation and has the therapeutic potential to prevent or treat oxidative stress-related liver injury.

Association of low dietary folate intake with lower CAMKK2 gene methylation, adiposity, and insulin resistance in obese subjects

Folate deficiency has been putatively implicated in the onset of diverse metabolic abnormalities, including insulin resistance, by altering epigenetic processes on key regulatory genes. The calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is involved in the regulation of critical metabolic processes such as adiposity and glucose homeostasis. This study hypothesized associations between low folate intakes and lower methylation levels of the CAMKK2 gene, with the presence of metabolic alterations in subjects with obesity. A cross-sectional ancillary study was conducted in obese subjects (n=47) from the RESMENA study (Spain). Fat mass was measured by dual-energy x-ray absorptiometry. Dietary intake and metabolic profile were assessed by validated methods. DNA methylation and gene expression in peripheral white blood cells were analyzed by microarray approaches. A total of 51 cytosine-phosphate-guanine sites were associated with folate intake (false discovery rate values < 0.0001), including one located in the 5′ untranslated region of the CAMKK2 gene (Illumina ID, cg16942632), which was selected and separately analyzed. Subjects with total folate intake lower than 300μg/d showed more fat mass (especially trunk fat), as well as statistically higher levels of glucose, insulin, homeostatic model assessment–insulin resistance (HOMA-IR) index, cortisol, and plasminogen activator inhibitor-1 than those consuming at least or more than 300μg/d. Of note, folate deficiency was related to lower CAMKK2 methylation. Interestingly, CAMKK2 methylation negatively correlated with the HOMA-IR index. Furthermore, CAMKK2 expression directly correlated with HOMA-IR values. In summary, this study suggests associations between low folate intakes, lower CAMKK2 gene methylation, and insulin resistance in obese individuals.

Calcium/calmodulin-dependent kinase kinase 2 regulates hematopoietic stem and progenitor cell regeneration

Hematopoietic stem and progenitor cells (HSPCs) are predominantly quiescent in adults, but proliferate in response to bone marrow (BM) injury. Here, we show that deletion of Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) promotes HSPC regeneration and hematopoietic recovery following radiation injury. Using Camkk2-enhanced green fluorescent protein (EGFP) reporter mice, we found that Camkk2 expression is developmentally regulated in HSPC. Deletion of Camkk2 in HSPC results in a significant downregulation of genes affiliated with the quiescent signature. Accordingly, HSPC from Camkk2 null mice have a high proliferative capability when stimulated in vitro in the presence of BM-derived endothelial cells. In addition, Camkk2 null mice are more resistant to radiation injury and show accelerated hematopoietic recovery, enhanced HSPC regeneration and ultimately a prolonged survival following sublethal or lethal total body irradiation. Mechanistically, we propose that CaMKK2 regulates the HSPC response to hematopoietic damage by coupling radiation signaling to activation of the anti-proliferative AMP-activated protein kinase. Finally, we demonstrated that systemic administration of the small molecule CaMKK2 inhibitor, STO-609, to irradiated mice enhanced HSPC recovery and improved survival. These findings identify CaMKK2 as an important regulator of HSPC regeneration and demonstrate CaMKK2 inhibition is a novel approach to promoting hematopoietic recovery after BM injury.

Akt activation by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (β) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G1/S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca2+i chelation or CaM inhibition. Ionomycin-induced Ca2+ influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells.

Impact of Genetic Variation on Human CaMKK2 Regulation by Ca2+-Calmodulin and Multisite Phosphorylation

The Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) is a key regulator of neuronal function and whole-body energy metabolism. Elevated CaMKK2 activity is strongly associated with prostate and hepatic cancers, whereas reduced CaMKK2 activity has been linked to schizophrenia and bipolar disease in humans. Here we report the functional effects of nine rare-variant point mutations that were detected in large-scale human genetic studies and cancer tissues, all of which occur close to two regulatory phosphorylation sites and the catalytic site on human CaMKK2. Four mutations (G87R, R139W, R142W and E268K) cause a marked decrease in Ca2+-independent autonomous activity, however S137L and P138S mutants displayed increased autonomous and Ca2+-CaM stimulated activities. Furthermore, the G87R mutant is defective in Thr85-autophosphorylation dependent autonomous activity, whereas the A329T mutation rendered CaMKK2 virtually insensitive to Ca2+-CaM stimulation. The G87R and R139W mutants behave as dominant-negative inhibitors of CaMKK2 signaling in cells as they block phosphorylation of the downstream substrate AMP-activated protein kinase (AMPK) in response to ionomycin. Our study provides insight into functionally disruptive, rare-variant mutations in human CaMKK2, which have the potential to influence risk and burden of disease associated with aberrant CaMKK2 activity in human populations carrying these variants.

Investigation of curcumin-mediated signalling pathways in head and neck squamous cell carcinoma

Objectives: Curcumin has been shown to exhibit anti-neoplastic effects. However, due to its poor bioavailability, the use of curcumin as an anti-cancer drug is limited. Thus, it is necessary to identify molecules as an alternative to curcumin that could serve as anti-cancer targets. In this study, we attempted to understand the underlying curcumin-mediated signalling pathways contributing to anti-neoplastic effects of curcumin. Methods: We carried out mass spectrometry-based phosphoproteomic analysis of head and neck cancer cell line, CAL 27, treated with and without curcumin to identify curcumin-mediated signalling pathways. Serine/threonine kinases were enriched using titanium dioxide. Results: This resulted in the identification of 5921 phosphopeptides corresponding to 1878 proteins. Of these, 275 and 183 phosphopeptides corresponding to 335 and 242 proteins (≥2.0-fold) were found to be hyper- and hypo-phosphorylated, respectively, in response to curcumin treatment. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a serine/threonine kinase, and its downstream target protein kinase AMP-activated non-catalytic subunit beta 1 (PRKAB1) were found to be hypo-phosphorylated when treated with curcumin. Further, silencing or inhibiting CaMKK2 resulted in decreased invasion and colony forming ability of not only CAL 27 cells but also other head and neck squamous cell carcinoma (HNSCC) cell lines. Further, Western blot analysis showed that curcumin-mediated signalling is corroborated by CaMKK2. Conclusions: Taken together, our results suggest that CaMKK2 could be a novel therapeutic target in HNSCC and can serve as an alternative to curcumin.

Abstract P214: The Non-canonical Stress Tolerance Cascade for Toll-like Receptor 9 in the Vasculature Signals Through Liver Kinase B1

Toll-like receptor (TLR)9 is a pattern recognition receptor of the innate immune system. Recently, a non-canonical stress tolerance pathway has been reported for TLR9 in non-immune cells (cardiomyocytes and neurons), independent of inflammatory signaling. It was observed that TLR9 inhibited sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA)2, increasing cytosolic calcium, and resulting in 5’ AMP-activated protein kinase (AMPK)α activation. In our laboratory, we have reported that TLR9 treatment in vivo causes arterial dysfunction that contributes to the pathogenesis of hypertension and that these phenotypes occurred in conjunction with vascular AMPKα phosphorylation (Thr172). However, whether a dysregulation in calcium homeostasis via the non-canonical stress tolerance cascade underlies the impaired vascular function after TLR9 stimulation needs to be clarified. We hypothesized that TLR9 activation would inhibit SERCA2 activity in the vasculature. SERCA2 activity was assessed using a luciferase-based ATP quantification kit. Microsomes were isolated from pooled aortae of Sprague-Dawley rats and subjected to treatment with either Vehicle (Veh) or ODN2395 (2 μM), with or without a SERCA2 inhibitor (thapsigargin; 1 μM). The presence of thapsigargin increased ATP concentrations similarly in both Veh and ODN2395 [ATP (μM), Veh: 19±3 vs. Veh+thapsigargin: 140±35; ODN2395: 22±9 vs. ODN2395+thapsigargin: 129±12, both p&lt;0.05], suggesting TLR9 activation did not inhibit SERCA2 activity. Next, MRA from Sprague-Dawley rats were divided into three sections for Western blot analysis of AMPKα-activating kinases, specifically calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and liver kinase B1 (LKB1). ODN2395 alone did not increase protein expression of phospho-CaMKK2 Ser511 (p&gt;0.05), again suggesting calcium-independent activation of AMPKα. However, ODN2395 did increase phospho-LKB1 Ser428 (3.8 fold vs. Veh, p&lt;0.05), and this increase in expression was inhibited by pre-incubation with TLR9 antagonist ODN2088 (20 μM) (p&gt;0.05). These results suggest that the TLR9 non-canonical stress tolerance pathway in the vasculature is mediated by LKB1, and not SERCA2 inhibition.

Micro-computed tomography assisted distal femur metaphyseal blunt punch compression for determining trabecular bone strength in mice

Shorter generation time and the power of genetic manipulation make mice an ideal model system to study bone biology as well as bone diseases. However their small size presents a challenge to perform strength measurements, particularly of the weight-bearing cancellous bone in the murine long bones. We recently developed an improved method to measure the axial compressive strength of the cancellous bone in the distal femur metaphysis in mice. Transverse micro-computed tomography image slices that are 7µm thick were used to locate the position where the epiphysis–metaphysis transition occurs. This enabled the removal of the distal femur epiphysis at the exact transition point exposing the full extent of metaphyseal trabecular bone, allowing more accurate and consistent measurement of its strength. When applied to a murine model system consisting of five month old male wild-type (WT) and Ca2+/calmodulin dependent protein kinase kinase 2 (CaMKK2) knockout (KO) Camkk2−/− mice that possess recorded differences in trabecular bone volume, data collected using this method showed good correlation between bone volume fraction and strength of trabecular bone. In combination with micro-computed tomography and histology, this method will provide a comprehensive and consistent assessment of the microarchitecture and tissue strength of the cancellous bone in murine mouse models.