Small Molecule Inhibition of the Autophagy Kinase ULK1 and Identification of ULK1 Substrates.

Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation, how ULK1 promotes autophagy remains poorly understood. Here, we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered 15 phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.

MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer.

While altered expression of microRNAs (miRs) in tumors has been well documented, it remains unclear how the miR transcriptome intersects neoplastic progression. By profiling the miR transcriptome we identified miR expression signatures associated with steps in tumorigenesis and the acquisition of hallmark capabilities in a prototypical mouse model of cancer. Metastases and a rare subset of primary tumors shared a distinct miR signature, implicating a discrete lineage for metastatic tumors. The miR-200 family is strongly down-regulated in metastases and met-like primary tumors, thereby relieving repression of the mesenchymal transcription factor Zeb1, which in turn suppresses E-cadherin. Treatment with a clinically approved angiogenesis inhibitor normalized angiogenic signature miRs in primary tumors, while altering expression of metastatic signature miRs similarly to liver metastases, suggesting their involvement in adaptive resistance to anti-angiogenic therapy via enhanced metastasis. Many of the miR changes associated with specific stages and hallmark capabilities in the mouse model are similarly altered in human tumors, including cognate pancreatic neuroendocrine tumors, implying a generality.

Genetic deletion of the desmosomal component desmoplakin promotes tumor microinvasion in a mouse model of pancreatic neuroendocrine carcinogenesis.

We used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to profile the transcriptome of pancreatic neuroendocrine tumors (PNET) that were either non-invasive or highly invasive, seeking to identify pro- and anti-invasive molecules. Expression of multiple components of desmosomes, structures that help maintain cellular adhesion, was significantly reduced in invasive carcinomas. Genetic deletion of one of these desmosomal components, desmoplakin, resulted in increased local tumor invasion without affecting tumor growth parameters in RT2 PNETs. Expression of cadherin 1, a component of the adherens junction adhesion complex, was maintained in these tumors despite the genetic deletion of desmoplakin. Our results demonstrate that loss of desmoplakin expression and resultant disruption of desmosomal adhesion can promote increased local tumor invasion independent of adherens junction status.

Polymorphic genetic control of tumor invasion in a mouse model of pancreatic neuroendocrine carcinogenesis.

Cancer is a disease subject to both genetic and environmental influences. In this study, we used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to identify a genetic locus that influences tumor progression to an invasive growth state. RT2 mice inbred into the C57BL/6 (B6) background develop both noninvasive pancreatic neuroendocrine tumors (PNET) and invasive carcinomas with varying degrees of aggressiveness. In contrast, RT2 mice inbred into the C3HeB/Fe (C3H) background are comparatively resistant to the development of invasive tumors, as are RT2 C3HB6(F1) hybrid mice. Using linkage analysis, we identified a 13-Mb locus on mouse chromosome 17 with significant linkage to the development of highly invasive PNETs. A gene residing in this locus, the anaplastic lymphoma kinase (Alk), was expressed at significantly lower levels in PNETs from invasion-resistant C3H mice compared with invasion-susceptible B6 mice, and pharmacological inhibition of Alk led to reduced tumor invasiveness in RT2 B6 mice. Collectively, our results demonstrate that tumor invasion is subject to polymorphic genetic control and identify Alk as a genetic modifier of invasive tumor growth.

The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non-small cell lung cancer.

The LKB1 tumor suppressor is often mutationally inactivated in non-small cell lung cancer (NSCLC). LKB1 phosphorylates and activates members of the AMPK family of Ser/Thr kinases. Within this family, the salt-inducible kinases (SIKs) modulate gene expression in part via the inhibitory phosphorylation of the CRTCs, coactivators for CREB (cAMP response element-binding protein). The loss of LKB1 causes SIK inactivation and the induction of the CRTCs, leading to the up-regulation of CREB target genes. We identified CRTC2 as a critical factor in LKB1-deficient NSCLC. CRTC2 is unphosphorylated and therefore constitutively activated in LKB1-mutant NSCLC, where it promotes tumor growth, in part via the induction of the inhibitor of DNA binding 1 (ID1), a bona fide CREB target gene. As ID1 expression is up-regulated and confers poor prognosis in LKB1-deficient NSCLC, our results suggest that small molecules that inhibit CRTC2 and ID1 activity may provide therapeutic benefit to individuals with NSCLC.