The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

Despite early studies linking calcium-calmodulin protein kinase kinase 2 (CAMKK2) to prostate cancer cell migration and invasion, the role of CAMKK2 in metastasis in vivo remains unclear. Moreover, while CAMKK2 is known to regulate systemic metabolism, whether CAMKK2’s effects on whole-body metabolism would impact prostate cancer progression and/or related comorbidities is not known. Here, we demonstrate that germline ablation of Camkk2 slows, but does not stop, primary prostate tumorigenesis in the TRansgenic Adenocarcinoma Mouse Prostate (TRAMP) genetic mouse model. Consistent with prior epidemiological reports supporting a link between obesity and prostate cancer aggressiveness, TRAMP mice fed a high-fat diet exhibited a pronounced increase in the colonization of lung metastases. We demonstrated that this effect on the metastatic spread was dependent on CAMKK2. Notably, diet-induced lung metastases exhibited a highly aggressive neuroendocrine phenotype. Concurrently, Camkk2 deletion improved insulin sensitivity in the same mice. Histological analyses revealed that cancer cells were smaller in the TRAMP;Camkk2^−/− mice compared to TRAMP;Camkk2^+/+ controls. Given the differences in circulating insulin levels, a known regulator of cell growth, we hypothesized that systemic CAMKK2 could promote prostate cancer cell growth and disease progression in part through cancer cell-extrinsic mechanisms. Accordingly, host deletion of Camkk2 impaired the growth of syngeneic murine prostate tumors in vivo, confirming nonautonomous roles for CAMKK2 in prostate cancer. Cancer cell size and mTOR signaling was diminished in tumors propagated in Camkk2-null mice. Together, these data indicate that, in addition to cancer cell-intrinsic roles, CAMKK2 mediates prostate cancer progression via tumor-extrinsic mechanisms. Further, we propose that CAMKK2 inhibition may also help combat common metabolic comorbidities in men with advanced prostate cancer.

Previous work has suggested androgen receptor (AR) signaling mediates cancer progression in part through the modulation of autophagy. Accordingly, we demonstrate that chloroquine, an inhibitor of autophagy, can inhibit tumor growth in preclinical mouse models of castration-resistant prostate cancer (CRPC). However, clinical trials testing chloroquine derivatives in men with CRPC have yet to yield promising results, potentially due to side effects. We hypothesized that identification of the upstream activators of autophagy in prostate cancer could highlight alternative, context-dependent targets for blocking this important cellular process during disease progression. Here, we used molecular (inducible overexpression and shRNA-mediated knockdown), genetic (CRISPR/Cas9), and pharmacological approaches to elucidate an AR-mediated autophagy cascade involving Ca ²⁺ /calmodulin-dependent protein kinase kinase 2 (CAMKK2; a kinase with a restricted expression profile), 5’-AMP-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase 1 (ULK1). These findings are consistent with data indicating CAMKK2-AMPK-ULK1 signaling correlates with disease progression in genetic mouse models and patient tumor samples. Importantly, CAMKK2 disruption impaired tumor growth and prolonged survival in multiple CRPC preclinical mouse models. Finally, we demonstrate that, similar to CAMKK2 inhibition, a recently described inhibitor of AMPK-ULK1 signaling blocked autophagy, cell growth and colony formation in prostate cancer cells. Taken together, our findings converge to demonstrate that AR signaling can co-opt the CAMKK2-AMPK-ULK1 signaling cascade to promote prostate cancer by increasing autophagy. Further, we propose that an inhibitor of this signaling cascade could serve as an alternative, more specific therapeutic compared to existing inhibitors of autophagy that, to date, have demonstrated limited efficacy in clinical trials due to their toxicity and poor pharmacokinetics.