Calcium/Calmodulin Dependent Protein Kinase Kinase 2 Regulates the Expansion of Tumor-Induced Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are a hetero geneous group of cells, which can suppress the immune response, promote tumor progression and impair the efficacy of immunotherapies. Consequently, the pharmacological targeting of MDSC is emerging as a new immunotherapeutic strategy to stimulate the natural anti-tumor immune response and potentiate the efficacy of immunotherapies. Herein, we leveraged genetically modified models and a small molecule inhibitor to validate Calcium-Calmodulin Kinase Kinase 2 (CaMKK2) as a druggable target to control MDSC accumulation in tumor-bearing mice. The results indicated that deletion of CaMKK2 in the host attenuated the growth of engrafted tumor cells, and this phenomenon was associated with increased antitumor T cell response and decreased accumulation of MDSC. The adoptive transfer of MDSC was sufficient to restore the ability of the tumor to grow in Camkk2-/- mice, confirming the key role of MDSC in the mechanism of tumor rejection. In vitro studies indicated that blocking of CaMKK2 is sufficient to impair the yield of MDSC. Surprisingly, MDSC generated from Camkk2-/- bone marrow cells also showed a higher ability to terminally differentiate toward more immunogenic cell types (e.g inflammatory macrophages and dendritic cells) compared to wild type (WT). Higher intracellular levels of reactive oxygen species (ROS) accumulated in Camkk2-/- MDSC, increasing their susceptibility to apoptosis and promoting their terminal differentiation toward more mature myeloid cells. Mechanistic studies indicated that AMP-activated protein kinase (AMPK), which is a known CaMKK2 proximal target controlling the oxidative stress response, fine-tunes ROS accumulation in MDSC. Accordingly, failure to activate the CaMKK2-AMPK axis can account for the elevated ROS levels in Camkk2-/- MDSC. These results highlight CaMKK2 as an important regulator of the MDSC lifecycle, identifying this kinase as a new druggable target to restrain MDSC expansion and enhance the efficacy of anti-tumor immunotherapy.

Genetic deletion of calcium/calmodulin-dependent protein kinase kinase ß (CaMKK ß) or CaMK IV exacerbates stroke outcomes in ovariectomized (OVXed) female mice.

BACKGROUND: Stroke is the primary cause of long-term disability in the United States. Interestingly, mounting evidence has suggested potential sex differences in the response to stroke treatment in patients as, at least in part, distinct cell death programs may be triggered in females and males following stroke. The NIH has recognized that females are strikingly under-represented in pre-clinical trials. Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is a major kinase that is activated by elevated intracellular calcium. It has recently been suggested that CaMKK and CaMK IV, a downstream target molecule, are neuroprotective in stroke in males. In this study, we examined stroke outcomes in ovariectomized CaMKK ß and CaMK IV deficient females. Cell death/survival signaling and inflammatory responses were assessed. RESULTS: Our results demonstrated that CaMKK ß or CaMK IV KO exacerbated both ischemic injury and behavioral deficits in female mice. Genetic deletion of CaMKK ß or CaMK IV increased hemorrhagic transformation after stroke, and this was associated with both increased MMP9 activity and loss of the blood brain barrier (BBB) protein collagen IV. Transcriptional inactivation was observed in mice lacking either CaMKK ß or CaMK IV, as indicated by reduced levels of phosphorylated cAMP response element-binding protein (p-CREB) and B-cell lymphoma 2 (BCL-2) proteins. Finally, inhibiting this pathway exacerbated the inflammatory response to stroke as CaMKK ß or CaMK IV KO mice had increased levels of the pro-inflammatory serum cytokines tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6) after stroke. This suggests that the CaMKK pathway is involved in the immune response to brain injury. CONCLUSIONS: Inhibition of CaMKK signaling exacerbated stroke outcome and increased BBB impairment, transcriptional inactivation and inflammatory responses in females after stroke. Therefore, CaMKK signaling may be a potential target for stroke treatment in both males and females.

Calcium/calmodulin-dependent protein kinase kinase 2 regulates macrophage-mediated inflammatory responses.

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) plays a key role in regulating food intake and energy expenditure at least in part by its actions in hypothalamic neurons. Previously, we showed that loss of CaMKK2 protected mice from high-fat diet (HFD)-induced obesity and glucose intolerance. However, although pair feeding HFD to WT mice to match food consumption of CAMKK2-null mice slowed weight gain, it failed to protect from glucose intolerance. Here we show that relative to WT mice, HFD-fed CaMKK2-null mice are protected from inflammation in adipose and remain glucose-tolerant. Moreover, loss of CaMKK2 also protected mice from endotoxin shock and fulminant hepatitis. We explored the expression of CaMKK2 in immune cells and found it to be restricted to those of the monocyte/macrophage lineage. CaMKK2-null macrophages exhibited a remarkable deficiency to spread, phagocytose bacteria, and synthesize cytokines in response to the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS). Mechanistically, loss of CaMKK2 uncoupled the TLR4 cascade from activation of protein tyrosine kinase 2 (PYK2; also known as PTK2B). Our findings uncover an important function for CaMKK2 in mediating mechanisms that control the amplitude of macrophage inflammatory responses to excess nutrients or pathogen derivatives.