Multi-omic dataset of patient-derived tumor organoids of neuroendocrine neoplasms.

BACKGROUND: Organoids are 3-dimensional experimental models that summarize the anatomical and functional structure of an organ. Although a promising experimental model for precision medicine, patient-derived tumor organoids (PDTOs) have currently been developed only for a fraction of tumor types. RESULTS: We have generated the first multi-omic dataset (whole-genome sequencing [WGS] and RNA-sequencing [RNA-seq]) of PDTOs from the rare and understudied pulmonary neuroendocrine tumors (n = 12; 6 grade 1, 6 grade 2) and provide data from other rare neuroendocrine neoplasms: small intestine (ileal) neuroendocrine tumors (n = 6; 2 grade 1 and 4 grade 2) and large-cell neuroendocrine carcinoma (n = 5; 1 pancreatic and 4 pulmonary). This dataset includes a matched sample from the parental sample (primary tumor or metastasis) for a majority of samples (21/23) and longitudinal sampling of the PDTOs (1 to 2 time points), for a total of n = 47 RNA-seq and n = 33 WGS. We here provide quality control for each technique and the raw and processed data as well as all scripts for genomic analyses to ensure an optimal reuse of the data. In addition, we report gene expression data and somatic small variant calls and describe how they were generated, in particular how we used WGS somatic calls to train a random forest classifier to detect variants in tumor-only RNA-seq. We also report all histopathological images used for medical diagnosis: hematoxylin and eosin-stained slides, brightfield images, and immunohistochemistry images of protein markers of clinical relevance. CONCLUSIONS: This dataset will be critical to future studies relying on this PDTO biobank, such as drug screens for novel therapies and experiments investigating the mechanisms of carcinogenesis in these understudied diseases.

Druggable growth dependencies and tumor evolution analysis in patient-derived organoids of neuroendocrine neoplasms from multiple body sites.

Neuroendocrine neoplasms (NENs) comprise well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Treatment options for patients with NENs are limited, in part due to lack of accurate models. We establish patient-derived tumor organoids (PDTOs) from pulmonary NETs and derive PDTOs from an understudied subtype of NEC, large cell neuroendocrine carcinoma (LCNEC), arising from multiple body sites. PDTOs maintain the gene expression patterns, intra-tumoral heterogeneity, and evolutionary processes of parental tumors. Through hypothesis-driven drug sensitivity analyses, we identify ASCL1 as a potential biomarker for response of LCNEC to treatment with BCL-2 inhibitors. Additionally, we discover a dependency on EGF in pulmonary NET PDTOs. Consistent with these findings, we find that, in an independent cohort, approximately 50% of pulmonary NETs express EGFR. This study identifies an actionable vulnerability for a subset of pulmonary NETs, emphasizing the utility of these PDTO models.

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.

PKM2 is not required for pancreatic ductal adenocarcinoma.

BACKGROUND: While most cancer cells preferentially express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2), PKM2 is dispensable for tumor development in several mouse cancer models. PKM2 is expressed in human pancreatic cancer, and there have been conflicting reports on the association of PKM2 expression and pancreatic cancer patient survival, but whether PKM2 is required for pancreatic cancer progression is unknown. To investigate the role of PKM2 in pancreatic cancer, we used a conditional allele to delete PKM2 in a mouse model of pancreatic ductal adenocarcinoma (PDAC). RESULTS: PDAC tumors were initiated in LSL-Kras G12D/+ ;Trp53 flox/flox ;Pdx-1-Cre (KP-/-C) mice harboring a conditional Pkm2 allele. Immunohistochemical analysis showed PKM2 expression in wild-type tumors and loss of PKM2 expression in tumors from Pkm2 conditional mice. PKM2 deletion had no effect on overall survival or tumor size. Loss of PKM2 resulted in pyruvate kinase M1 (PKM1) expression, but did not affect the number of proliferating cells. These findings are consistent with results in other cancer models. CONCLUSIONS: PKM2 is not required for initiation or growth of PDAC tumors arising in the KP-/-C pancreatic cancer model. These findings suggest that, in this mouse PDAC model, PKM2 expression is not required for pancreatic tumor formation or progression.

Isoform-specific deletion of PKM2 constrains tumor initiation in a mouse model of soft tissue sarcoma.

BACKGROUND: Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of the glycolytic enzyme pyruvate kinase. PKM2 expression is associated with embryogenesis, tissue regeneration, and cancer. PKM2 is also the pyruvate kinase isoform expressed in most wild-type adult tissues, with PKM1 restricted primarily to skeletal muscle, heart, and brain. To interrogate the functional requirement for PKM2 during tumor initiation in an autochthonous mouse model for soft tissue sarcoma (STS), we used a conditional Pkm2 allele (Pkm2fl ) to abolish PKM2 expression. RESULTS: PKM2 deletion slowed tumor onset but did not abrogate eventual tumor outgrowth. PKM2-null sarcoma cells expressed PKM1 with tumors containing a high number of infiltrating PKM2 expressing stromal cells. End-stage PKM2-null tumors showed increased proliferation compared to tumors with a wild-type Pkm2 allele, and tumor metabolite analysis revealed metabolic changes associated with PKM2 loss. CONCLUSIONS: While PKM2 is not required for soft tissue sarcoma growth, PKM2 expression may facilitate initiation of this tumor type. Because these data differ from what has been observed in other cancer models where PKM2 has been deleted, they argue that the consequences of PKM2 loss during tumor initiation are dependent on the tumor type.

PKM2 is not required for colon cancer initiated by APC loss.

BACKGROUND: Cancer cells express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2). PKM2 expression is not required for some cancers, and PKM2 loss can promote cancer progression; however, PKM2 has been reported to be essential in other tumor contexts, including a proposed non-metabolic role in ß-catenin nuclear translocation. PKM2 is expressed in colon cancers where loss of the Apc tumor suppressor results in ß-catenin nuclear translocation and aberrant activation of the canonical Wnt signaling pathway. Whether PKM2 is required in this colon cancer context has not been investigated. RESULTS: Colon tumorigenesis was induced in mice harboring conditional Apc and Pkm2 alleles, and tumor progression was monitored by serial colonoscopy. PKM2 deletion had no effect on overall survival, the number of mice that developed tumors, or the number of tumors that developed per animal. Immunohistochemical analysis demonstrated PKM2 expression in wild-type tumors and the expected loss of PKM2 expression in tumors from Pkm2 conditional mice. Loss of PKM2 resulted in pyruvate kinase M1 expression but had no effect on nuclear ß-catenin staining. These findings are consistent with tumor growth and activated Wnt signaling despite PKM2 loss in this model. We also found a large fraction of human colon cancers had very low or undetectable levels of PKM2 expression. CONCLUSIONS: PKM2 is not required for Apc-deficient colon cancer or for nuclear translocation of ß-catenin in Apc-null tumor cells. These findings suggest that PKM2 expression is not required for colon tumor formation or progression.

Quantitative proteomics identify Tenascin-C as a promoter of lung cancer progression and contributor to a signature prognostic of patient survival.

The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients.

PKM2, cancer metabolism, and the road ahead.

A major metabolic aberration associated with cancer is a change in glucose metabolism. Isoform selection of the glycolytic enzyme pyruvate kinase has been implicated in the metabolic phenotype of cancer cells, and specific pyruvate kinase isoforms have been suggested to support divergent energetic and biosynthetic requirements of cells in tumors and normal tissues. PKM2 isoform expression has been closely linked to embryogenesis, tissue repair, and cancer. In contrast, forced expression of the PKM1 isoform has been associated with reduced tumor cell proliferation. Here, we discuss the role that PKM2 plays in cells and provide a historical perspective for how the study of PKM2 has contributed to understanding cancer metabolism. We also review recent studies that raise important questions with regard to the role of PKM2 in both normal and cancer cell metabolism.

Germline loss of PKM2 promotes metabolic distress and hepatocellular carcinoma.

Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase (PK), and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. To interrogate the functional requirement for PKM2 during development and tissue homeostasis, we generated germline PKM2-null mice (Pkm2(-/-)). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2(-/-) mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. Strikingly, PKM2 loss leads to spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism.

Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation.

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.

Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development.

Most patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed with advanced disease and survive less than 12 months. PDAC has been linked with obesity and glucose intolerance, but whether changes in circulating metabolites are associated with early cancer progression is unknown. To better understand metabolic derangements associated with early disease, we profiled metabolites in prediagnostic plasma from individuals with pancreatic cancer (cases) and matched controls from four prospective cohort studies. We find that elevated plasma levels of branched-chain amino acids (BCAAs) are associated with a greater than twofold increased risk of future pancreatic cancer diagnosis. This elevated risk was independent of known predisposing factors, with the strongest association observed among subjects with samples collected 2 to 5 years before diagnosis, when occult disease is probably present. We show that plasma BCAAs are also elevated in mice with early-stage pancreatic cancers driven by mutant Kras expression but not in mice with Kras-driven tumors in other tissues, and that breakdown of tissue protein accounts for the increase in plasma BCAAs that accompanies early-stage disease. Together, these findings suggest that increased whole-body protein breakdown is an early event in development of PDAC.

PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells.

The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.

Differential Tks5 isoform expression contributes to metastatic invasion of lung adenocarcinoma.

Metastasis accounts for the vast majority of cancer-related deaths, yet the molecular mechanisms that drive metastatic spread remain poorly understood. Here we report that Tks5, which has been linked to the formation of proteolytic cellular protrusions known as invadopodia, undergoes an isoform switch during metastatic progression in a genetically engineered mouse model of lung adenocarcinoma. Nonmetastatic primary tumor-derived cells predominantly expressed a short isoform, Tks5short, while metastatic primary tumor- and metastasis-derived cells acquired increased expression of the full-length isoform Tks5long. This elevation of Tks5long to Tks5short ratio correlated with a commensurate increase in invadopodia activity in metastatic cells compared with nonmetastatic cells. Further characterization of these isoforms by knockdown and overexpression experiments demonstrated that Tks5long promoted invadopodia in vitro and increased metastasis in transplant models and an autochthonous model of lung adenocarcinoma. Conversely, Tks5short decreased invadopodia stability and proteolysis, acting as a natural dominant-negative inhibitor to Tks5long. Importantly, high Tks5long and low Tks5short expressions in human lung adenocarcinomas correlated with metastatic disease and predicted worse survival of early stage patients. These data indicate that tipping the Tks5 isoform balance to a high Tks5long to Tks5short ratio promotes invadopodia-mediated invasion and metastasis.

Nuclear factor I/B is an oncogene in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive cancer often diagnosed after it has metastasized. Despite the need to better understand this disease, SCLC remains poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.

Suppression of lung adenocarcinoma progression by Nkx2-1.

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras(LSL-G12D/+);p53(flox/flox) mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.