Supplementary Figure from Pyruvate Kinase M1 Suppresses Development and Progression of Prostate Adenocarcinoma

Abstract Altered metabolism helps sustain cancer cell proliferation and survival. Most cancers, including prostate cancers, express the M2 splice isoform of pyruvate kinase (PKM2), which can support anabolic metabolism to support cell proliferation. However, Pkm2 expression is dispensable for the formation and growth of many cancers in vivo. Expression of pyruvate kinase isoform M1 (Pkm1) is restricted to relatively few tissues and has been reported to promote growth of select tumors, but the role of PKM1 in cancer has been less studied than PKM2. To test how differential expression of pyruvate kinase isoforms affects cancer initiation and progression, we generated mice harboring a conditional allele of Pkm1 and crossed these mice, or those with a Pkm2 conditional allele, with a Pten loss-driven prostate cancer model. Pkm1 loss led to increased PKM2 expression and accelerated prostate cancer development, whereas Pkm2 deletion led to increased PKM1 expression and suppressed tumor progression. Metabolic profiling revealed altered nucleotide levels in tumors with high PKM1 expression, and failure of these tumors to progress was associated with DNA replication stress and senescence. Consistent with these data, a small molecule pyruvate kinase activator that mimics a high activity PKM1-like state suppressed progression of established prostate tumors. Analysis of human specimens showed PKM2 expression is retained in most human prostate cancers. Overall, this study uncovers a role for pyruvate kinase isoforms in prostate cancer initiation and progression, and argues that pharmacologic pyruvate kinase activation may be beneficial for treating prostate cancer. Significance: Differential expression of PKM1 and PKM2 impacts prostate tumorigenesis and suggests a potential therapeutic vulnerability in prostate cancer.

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.

Defining the metabolic limitations of tumour growth will help to develop cancer therapies(1). 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 impaired(2-4); 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 asparagine(5), 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.

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.

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.

Nat. Chem. Biol. 8, 839–847 (2012); published online 26 August 2012; corrected after print 11 October 2012 In the version of this article initially published, the accession codes to the Protein Data Bank were omitted. The error has been corrected in the HTML and PDF versions of the article.