Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing.

Small cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally, we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs.

Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer.

BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis.

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

Pyruvate Kinase M1 Suppresses Development and Progression of Prostate Adenocarcinoma.

SIGNIFICANCE: Differential expression of PKM1 and PKM2 impacts prostate tumorigenesis and suggests a potential therapeutic vulnerability in prostate cancer.

Stabilization of the Max Homodimer with a Small Molecule Attenuates Myc-Driven Transcription.

The transcription factor Max is a basic-helix-loop-helix leucine zipper (bHLHLZ) protein that forms homodimers or interacts with other bHLHLZ proteins, including Myc and Mxd proteins. Among this dynamic network of interactions, the Myc/Max heterodimer has crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Despite this significance, the arsenal of high-quality chemical probes to interrogate these proteins remains limited. We used small molecule microarrays to identify compounds that bind Max in a mechanistically unbiased manner. We discovered the asymmetric polycyclic lactam, KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppresses tumor growth in vivo. This approach demonstrates the feasibility of modulating Max with small molecules and supports altering Max dimerization as an alternative approach to targeting Myc.

Aspartate is an endogenous metabolic limitation for tumour growth.

Defining the metabolic limitations of tumour growth will help to develop cancer therapies1. Cancer cells proliferate slower in tumours than in standard culture conditions, indicating that a metabolic limitation may restrict cell proliferation in vivo. Aspartate synthesis can limit cancer cell proliferation when respiration is impaired2-4; however, whether acquiring aspartate is endogenously limiting for tumour growth is unknown. We confirm that aspartate has poor cell permeability, which prevents environmental acquisition, whereas the related amino acid asparagine is available to cells in tumours, but cancer cells lack asparaginase activity to convert asparagine to aspartate. Heterologous expression of guinea pig asparaginase 1 (gpASNase1), an enzyme that produces aspartate from asparagine5, confers the ability to use asparagine to supply intracellular aspartate to cancer cells in vivo. Tumours expressing gpASNase1 grow at a faster rate, indicating that aspartate acquisition is an endogenous metabolic limitation for the growth of some tumours. Tumours expressing gpASNase1 are also refractory to the growth suppressive effects of metformin, suggesting that metformin inhibits tumour growth by depleting aspartate. These findings suggest that therapeutic aspartate suppression could be effective to treat cancer.

In situ pulmonary localization of air pollution particle-induced oxidative stress.

Exposure to air particulate matter (PM) may be associated with increased morbidity and mortality. An improved understanding of the mechanism(s) by which PM induces adverse effects is needed. This preliminary study examined the ability to use unique bioluminescent technologies to identify acute localized areas of residual oil fly ash (ROFA)-induced, oxidative lung injury. Transgenic mice, in which luciferase (luc) expression was regulated by the heme oxygenase (HO)-1 promoter, were exposed by pharyngeal aspiration to either saline or 50 microg ROFA/mouse. HO-1-luc expression was determined at 2, 6, 12, and 24 h postexposure using luminescent quantification and Western blot analysis of lung protein extracts, as well as with a novel in situ pulmonary bioluminescence imaging approach. The different approaches for the detection of luciferase in lung protein extracts recovered from ROFA exposed HO-1-luc transgenic mice gave variable results. Pulmonary homogenate HO-1-luc levels were increased at 2 h and 24 h postexposure to ROFA when examined by chemilumescent and Western blot analyses, respectively. In situ bioluminescent imaging of pulmonary tissue sections detected ROFA-induced pulmonary luciferase expression by identifying highly localized increases in HO-1-luc expression at 12 h and 24 h postexposure. These results suggest that the variability observed in the methods of detection for luciferase may be due to a localization of cells expressing luciferase within tissue samples, demonstrating that the HO-1-luc transgenic mouse model is the preferred method to detect and pinpoint in vivo particle-induced, oxidative lung injury. The feasibility of using this in situ approach is a unique proof-of-concept application for the identification of localized sites of oxidative injury induced by environmental pollutants.