HDAC3 is critical in tumor development and therapeutic resistance in Kras-mutant non-small cell lung cancer.

HDAC3 is one of the main targets of histone deacetylase (HDAC) inhibitors in clinical development as cancer therapies, yet the in vivo role of HDAC3 in solid tumors is unknown. We identified a critical role for HDAC3 in Kras-mutant lung cancer. Using genetically engineered mouse models (GEMMs), we found that HDAC3 is required for lung tumor growth in vivo. HDAC3 was found to direct and enhance the transcription effects of the lung cancer lineage transcription factor NKX2-1 to mediate expression of a common set of target genes. We identified FGFR1 as a critical previously unidentified target of HDAC3. Leveraging this, we identified that an HDAC3-dependent transcriptional cassette becomes hyperactivated as Kras/LKB1-mutant cells develop resistance to the MEK inhibitor trametinib, and this can be reversed by treatment with the HDAC1/HDAC3 inhibitor entinostat. We found that the combination of entinostat plus trametinib treatment elicits therapeutic benefit in the Kras/LKB1 GEMM.

AMPK/ULK1-mediated phosphorylation of Parkin ACT domain mediates an early step in mitophagy.

The serine/threonine kinase ULK1 mediates autophagy initiation in response to various cellular stresses, and genetic deletion of ULK1 leads to accumulation of damaged mitochondria. Here we identify Parkin, the core ubiquitin ligase in mitophagy, and PARK2 gene product mutated in familial Parkinson's disease, as a ULK1 substrate. Recent studies uncovered a nine residue ("ACT") domain important for Parkin activation, and we demonstrate that AMPK-dependent ULK1 rapidly phosphorylates conserved serine108 in the ACT domain in response to mitochondrial stress. Phosphorylation of Parkin Ser108 occurs maximally within five minutes of mitochondrial damage, unlike activation of PINK1 and TBK1, which is observed thirty to sixty minutes later. Mutation of the ULK1 phosphorylation sites in Parkin, genetic AMPK or ULK1 depletion, or pharmacologic ULK1 inhibition, all lead to delays in Parkin activation and defects in assays of Parkin function and downstream mitophagy events. These findings reveal an unexpected first step in the mitophagy cascade.

Inhibition of CAMKK2 impairs autophagy and castration-resistant prostate cancer via suppression of AMPK-ULK1 signaling.

Previous work has suggested androgen receptor (AR) signaling mediates prostate cancer progression in part through the modulation of autophagy. However, clinical trials testing autophagy inhibition using chloroquine derivatives in men with castration-resistant prostate cancer (CRPC) have yet to yield promising results, potentially due to the side effects of this class of compounds. 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, genetic, and pharmacological approaches to elucidate an AR-mediated autophagy cascade involving Ca2+/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), but independent of canonical mechanistic target of rapamycin (mTOR) activity. Increased CAMKK2-AMPK-ULK1 signaling correlated 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. Similarly, an inhibitor of AMPK-ULK1 blocked autophagy, cell growth, and colony formation in prostate cancer cells. Collectively, our findings converge to demonstrate that AR can co-opt the CAMKK2-AMPK-ULK1 signaling cascade to promote prostate cancer by increasing autophagy. Thus, this pathway may represent an alternative autophagic target in CRPC.

Design, Synthesis, and Characterization of an Orally Active Dual-Specific ULK1/2 Autophagy Inhibitor that Synergizes with the PARP Inhibitor Olaparib for the Treatment of Triple-Negative Breast Cancer.

Inhibition of autophagy, the major cellular recycling pathway in mammalian cells, is a promising strategy for the treatment of triple-negative breast cancer (TNBC). We previously reported SBI-0206965, a small molecule inhibitor of unc-51-like autophagy activating kinase 1 (ULK1), which is a key regulator of autophagy initiation. Herein, we describe the design, synthesis, and characterization of new dual inhibitors of ULK1 and ULK2 (ULK1/2). One inhibitor, SBP-7455 (compound 26), displayed improved binding affinity for ULK1/2 compared with SBI-0206965, potently inhibited ULK1/2 enzymatic activity in vitro and in cells, reduced the viability of TNBC cells and had oral bioavailability in mice. SBP-7455 inhibited starvation-induced autophagic flux in TNBC cells that were dependent on autophagy for survival and displayed synergistic cytotoxicity with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib against TNBC cells. These data suggest that combining ULK1/2 and PARP inhibition may have clinical utility for the treatment of TNBC.

The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive Effects of LKB1 in NSCLC.

Mutations in the LKB1 (also known as STK11) tumor suppressor are the third most frequent genetic alteration in non-small cell lung cancer (NSCLC). LKB1 encodes a serine/threonine kinase that directly phosphorylates and activates 14 AMPK family kinases ("AMPKRs"). The function of many of the AMPKRs remains obscure, and which are most critical to the tumor-suppressive function of LKB1 remains unknown. Here, we combine CRISPR and genetic analysis of the AMPKR family in NSCLC cell lines and mouse models, revealing a surprising critical role for the SIK subfamily. Conditional genetic loss of Sik1 revealed increased tumor growth in mouse models of Kras-dependent lung cancer, which was further enhanced by loss of the related kinase Sik3. As most known substrates of the SIKs control transcription, gene-expression analysis was performed, revealing upregulation of AP1 and IL6 signaling in common between LKB1- and SIK1/3-deficient tumors. The SIK substrate CRTC2 was required for this effect, as well as for proliferation benefits from SIK loss. SIGNIFICANCE: The tumor suppressor LKB1/STK11 encodes a serine/threonine kinase frequently inactivated in NSCLC. LKB1 activates 14 downstream kinases in the AMPK family controlling growth and metabolism, although which kinases are critical for LKB1 tumor-suppressor function has remained an enigma. Here we unexpectedly found that two understudied kinases, SIK1 and SIK3, are critical targets in lung cancer.This article is highlighted in the In This Issue feature, p. 1469.

Genetic Analysis Reveals AMPK Is Required to Support Tumor Growth in Murine Kras-Dependent Lung Cancer Models.

AMPK, a conserved sensor of low cellular energy, can either repress or promote tumor growth depending on the context. However, no studies have examined AMPK function in autochthonous genetic mouse models of epithelial cancer. Here, we examine the role of AMPK in murine KrasG12D-mediated non-small-cell lung cancer (NSCLC), a cancer type in humans that harbors frequent inactivating mutations in the LKB1 tumor suppressor-the predominant upstream activating kinase of AMPK and 12 related kinases. Unlike LKB1 deletion, AMPK deletion in KrasG12D lung tumors did not accelerate lung tumor growth. Moreover, deletion of AMPK in KrasG12D p53f/f tumors reduced lung tumor burden. We identified a critical role for AMPK in regulating lysosomal gene expression through the Tfe3 transcription factor, which was required to support NSCLC growth. Thus, AMPK supports the growth of KrasG12D-dependent lung cancer through the induction of lysosomes, highlighting an unrecognized liability of NSCLC.

Autophagy in Cancer: Regulation by Small Molecules.

During times of stress, autophagy is a cellular process that enables cells to reclaim damaged components by a controlled recycling pathway. This mechanism for cellular catabolism is dysregulated in cancer, with evidence indicating that cancer cells rely on autophagy in the hypoxic and nutrient-poor microenvironment of solid tumors. Mounting evidence suggests that autophagy has a role in the resistance of tumors to standard-of-care (SOC) therapies. Therefore, there is significant interest in the discovery of small molecules that can safely modulate autophagy. In this review, we describe recent advances in the identification of new pharmacological compounds that modulate autophagy, with a focus on their mode of action, value as probe compounds, and validation as potential therapeutics.

Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical models.

Continuous de novo fatty acid synthesis is a common feature of cancer that is required to meet the biosynthetic demands of a growing tumor. This process is controlled by the rate-limiting enzyme acetyl-CoA carboxylase (ACC), an attractive but traditionally intractable drug target. Here we provide genetic and pharmacological evidence that in preclinical models ACC is required to maintain the de novo fatty acid synthesis needed for growth and viability of non-small-cell lung cancer (NSCLC) cells. We describe the ability of ND-646-an allosteric inhibitor of the ACC enzymes ACC1 and ACC2 that prevents ACC subunit dimerization-to suppress fatty acid synthesis in vitro and in vivo. Chronic ND-646 treatment of xenograft and genetically engineered mouse models of NSCLC inhibited tumor growth. When administered as a single agent or in combination with the standard-of-care drug carboplatin, ND-646 markedly suppressed lung tumor growth in the Kras;Trp53-/- (also known as KRAS p53) and Kras;Stk11-/- (also known as KRAS Lkb1) mouse models of NSCLC. These findings demonstrate that ACC mediates a metabolic liability of NSCLC and that ACC inhibition by ND-646 is detrimental to NSCLC growth, supporting further examination of the use of ACC inhibitors in oncology.

WNT signaling increases proliferation and impairs differentiation of stem cells in the developing cerebellum.

The WNT pathway plays multiple roles in neural development and is crucial for establishment of the embryonic cerebellum. In addition, WNT pathway mutations are associated with medulloblastoma, the most common malignant brain tumor in children. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. Here we investigate the effects of canonical WNT signaling on two important classes of progenitors in the developing cerebellum: multipotent neural stem cells (NSCs) and granule neuron precursors (GNPs). We show that WNT pathway activation in vitro promotes proliferation of NSCs but not GNPs. Moreover, mice that express activated ß-catenin in the cerebellar ventricular zone exhibit increased proliferation of NSCs in that region, whereas expression of the same protein in GNPs impairs proliferation. Although ß-catenin-expressing NSCs proliferate they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation.

N-myc alters the fate of preneoplastic cells in a mouse model of medulloblastoma.

Studying the early stages of cancer can provide important insight into the molecular basis of the disease. We identified a preneoplastic stage in the patched (ptc) mutant mouse, a model for the brain tumor medulloblastoma. Preneoplastic cells (PNCs) are found in most ptc mutants during early adulthood, but only 15% of these animals develop tumors. Although PNCs are found in mice that develop tumors, the ability of PNCs to give rise to tumors has never been demonstrated directly, and the fate of cells that do not form tumors remains unknown. Using genetic fate mapping and orthotopic transplantation, we provide definitive evidence that PNCs give rise to tumors, and show that the predominant fate of PNCs that do not form tumors is differentiation. Moreover, we show that N-myc, a gene commonly amplified in medulloblastoma, can dramatically alter the fate of PNCs, preventing differentiation and driving progression to tumors. Importantly, N-myc allows PNCs to grow independently of hedgehog signaling, making the resulting tumors resistant to hedgehog antagonists. These studies provide the first direct evidence that PNCs can give rise to tumors, and demonstrate that identification of genetic changes that promote tumor progression is critical for designing effective therapies for cancer.