Gary Bellinger

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses

The glycolytic metabolism of cancers differs from normal tissues, allowing tumor cells to survive under oxidative stress. Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.

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.

Identification of CDCP1 as a hypoxia-inducible factor 2α (HIF-2α) target gene that is associated with survival in clear cell renal cell carcinoma patients

CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that is highly expressed in stem cells and frequently overexpressed and tyrosine-phosphorylated in cancer. CDCP1 promotes cancer cell metastasis. However, the mechanisms that regulate CDCP1 are not well-defined. Here we show that hypoxia induces CDCP1 expression and tyrosine phosphorylation in hypoxia-inducible factor (HIF)-2α–, but not HIF-1α–, dependent fashion. shRNA knockdown of CDCP1 impairs cancer cell migration under hypoxic conditions, whereas overexpression of HIF-2α promotes the growth of tumor xenografts in association with enhanced CDCP1 expression and tyrosine phosphorylation. Immunohistochemistry analysis of tissue microarray samples from tumors of patients with clear cell renal cell carcinoma shows that increased CDCP1 expression correlates with decreased overall survival. Together, these data support a critical role for CDCP1 as a unique HIF-2α target gene involved in the regulation of cancer metastasis, and suggest that CDCP1 is a biomarker and potential therapeutic target for metastatic cancers.

Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion

Significance Mutations in the PI3K pathway are highly prevalent in cancers, and isoform-specific and pan-PI3K inhibitors have entered clinical trials in both solid and hematologic malignancies. The PI3K δ-specific inhibitor idelalisib (in combination with rituximab) was recently approved for the treatment of chronic lymphocytic leukemia. However, identifying tumor types and biological mechanisms that predict for response to PI3K inhibitors as single agents or in combination has been a challenge. Our data indicate that PI3K inhibitors induce DNA damage in tumors that have defects in DNA damage-repair pathways and that they do so by impairing the production of Rib phosphate and amino acids needed for deoxynucleotide synthesis. We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1f/fp53f/f), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors.

Demethylation therapy as a targeted treatment for human papilloma virus-associated head and neck cancer

Purpose: DNA methylation in human papillomavirus–associated (HPV+) head and neck squamous cell carcinoma (HNSCC) may have importance for continuous expression of HPV oncogenes, tumor cell proliferation, and survival. Here, we determined activity of a global DNA-demethylating agent, 5-azacytidine (5-aza), against HPV+ HNSCC in preclinical models and explored it as a targeted therapy in a window trial enrolling patients with HPV+ HNSCC. Experimental Design: Sensitivity of HNSCC cells to 5-aza treatment was determined, and then 5-aza activity was tested in vivo using xenografted tumors in a mouse model. Finally, tumor samples from patients enrolled in a window clinical trial were analyzed to identify activity of 5-aza therapy in patients with HPV+ HNSCC. Results: Clinical trial and experimental data show that 5-aza induced growth inhibition and cell death in HPV+ HNSCC. 5-aza reduced expression of HPV genes, stabilized p53, and induced p53-dependent apoptosis in HNSCC cells and tumors. 5-aza repressed expression and activity of matrix metalloproteinases (MMP) in HPV+ HNSCC, activated IFN response in some HPV+ head and neck cancer cells, and inhibited the ability of HPV+ xenografted tumors to invade mouse blood vessels. Conclusions: 5-aza may provide effective therapy for HPV-associated HNSCC as an alternative or complement to standard cytotoxic therapy. Clin Cancer Res; 23(23); 7276–87. ©2017 AACR.

Loss of LZAP inactivates p53 and regulates sensitivity of cells to DNA damage in a p53-dependent manner

Chemotherapy and radiation, the two most common cancer therapies, exert their anticancer effects by causing damage to cellular DNA. However, systemic treatment damages DNA not only in cancer, but also in healthy cells, resulting in the progression of serious side effects and limiting efficacy of the treatment. Interestingly, in response to DNA damage, p53 seems to play an opposite role in normal and in the majority of cancer cells—wild-type p53 mediates apoptosis in healthy tissues, attributing to the side effects, whereas mutant p53 often is responsible for acquired cancer resistance to the treatment. Here, we show that leucine zipper-containing ARF-binding protein (LZAP) binds and stabilizes p53. LZAP depletion eliminates p53 protein independently of its mutation status, subsequently protecting wild-type p53 cells from DNA damage-induced cell death, while rendering cells expressing mutant p53 more sensitive to the treatment. In human non-small-cell lung cancer, LZAP levels correlated with p53 levels, suggesting that loss of LZAP may represent a novel mechanism of p53 inactivation in human cancer. Our studies establish LZAP as a p53 regulator and p53-dependent determinative of cell fate in response to DNA damaging treatment.

Abstract 4588: Identification of CDCP1 as a HIF-2α target gene involved in the regulation of cancer cell migration and metastasis.

CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that is highly expressed in stem cells and frequently overexpressed and tyrosine phosphorylated in cancer. CDCP1 promotes cancer cell metastasis. However, the mechanisms that regulate CDCP1 are not well defined. Studies from our laboratory revealed a biochemical pathway by which CDCP1 participates in the activation of Src-family kinase (SFK) members and the coupling of SFK-activation to the phosphorylation and regulation of protein kinase C-delta (PKC-δ). Here we show that hypoxia induces CDCP1 expression and tyrosine phosphorylation in a HIF-2α, but not HIF-1α, dependent fashion. shRNA knockdown of CDCP1 impairs cancer cell migration under hypoxic conditions, while overexpression of HIF-2α promotes the growth of tumor xenografts in association with enhanced CDCP1 expression and tyrosine phosphorylation, as well as, significantly promotes lung metastases in NOD/SCID mice. To investigate the relationship between HIF-2α and CDCP1 expression, we performed a correlation analysis in the largest up-to-date collection (Sanger Cell Line Project) of cancer cell line microarray data (n=732). We found a dramatic concordance in the expression of HIF-2α and CDCP1 (Pearson9s correlation, P Supported by NIH grant 5R01GM056203-15 to L.C.C and Dana Farber/Harvard Cancer Center Career Development Award to B.M.E. Citation Format: Brooke M. Emerling, Cyril Benes, Eric Bell, George Poulogiannis, Kevin Courtney, Hui Lui, Rayman Choo-Wing, Gary Bellinger, Stephen Soltoff, Lewis Cantley. Identification of CDCP1 as a HIF-2α target gene involved in the regulation of cancer cell migration and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4588. doi:10.1158/1538-7445.AM2013-4588

Abstract LB-365: Effective use of PI3K/MTOR and MEK inhibitors prior to hormone ablative therapy in PTEN-loss driven murine prostate cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The PTEN and p53 tumor suppressors are the most commonly altered genes in human cancer, including prostate cancer (PCa). Loss of PTEN is associated with increased Gleason score and clinical recurrence, and the majority of human metastatic PCas have PTEN loss via multiple mechanisms. Mice with prostate-specific homozygous deletion of PTEN develop invasive PCa albeit with prolonged latency of 6-8 months. Combined PTEN/p53 inactivation in mouse prostate elicits invasive cancer by 9 weeks of age and invariable lethality by 6 months of age. Since PTEN loss results in PI3K/mTOR pathway activation, we evaluated the impact of GSK458 (PI3K/mTOR inhibitor) and GSK418 (PI3K beta/delta isoform-specific inhibitor), singly and in combination with GSK212 (MEK inhibitor), in uncastrated, prostate-specific PTEN/p53 double knockout mice (4-6 months) and PTEN mice (11-14 months), respectively, harboring advanced PCa. The drugs were administered by daily oral gavage for 3 weeks with serial 18FDG-PET/MRI imaging at baseline, 2 days, 1 week, 2 weeks and 3 weeks post-treatment respectively. GSK458 treatment of PTEN/p53 and PTEN mice results in reduction in FDG-PET uptake as early as 24 h post-treatment, with 40% tumor shrinkage by 1 week post-treatment, but rapid regrowth of 18FDG-avid tumor by 2-3 weeks post-treatment. This acquired resistance was found to be mediated in part through upregulation of multiple receptor tyrosine kinases. In contrast, PTEN/p53 and PTEN mice did not respond to GSK418, by either FDG-PET or MRI analysis. We observed increased pERK/total ERK ratio by Western blot analysis and increased p-ERK staining by immunohistochemistry in GSK458 and GSK418-treated PTEN/53 mice, respectively. Treatment of PTEN/p53 and PTEN mice with GSK212 resulted in an approx. 40% reduction in tumor volume over 3 weeks. Treatment of PTEN/p53 mice with a combination of GSK458 plus GSK212 resulted in approx. 60% reduction of tumor volume at 3 week post-treatment. Strikingly, treatment of prostate-specific PTEN only mice with GSK458 plus GSK212 combination resulted in a >90% tumor regression at 3 weeks post-treatment. These results demonstrate the potential utlitity of PI3K/MEK-directed combination therapies in the neoadjuvant setting for locally advanced disease or hormone-sensitive phase in metastatic disease, thus delaying the need for hormone ablative therapy and its associated morbidity in advanced PCa. The data underscore the value of genetically engineered mouse models to co-clinically evaluate biomarkers of response and resistance to targeted therapies, and elucidate mechanisms of acquired resistance early in clinical development. The design of “personalized” combination therapies to overcome resistance to PI3K/MEK-directed therapies in the hormone-naive and castration-resistant contexts are currently underway in multiple GEMMs and Phase Ib co-clinical trials in advanced PCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-365. doi:1538-7445.AM2012-LB-365

Abstract LB-418: Targeting the PI3K/mTOR pathway in genetically engineered mouse models of prostate cancer

The PTEN and p53 tumor suppressors are among the most commonly inactivated or mutated genes in human cancer, including prostate cancer. Loss of PTEN is associated with increased pathologic Gleason score and risk of clinical recurrence, and 20-60% of human metastatic prostate cancers have loss of heterozygosity at the PTEN locus, resulting in hyperactivation of the PI3K/mTOR pathway. Mice with germline heterozygosity for PTEN have been shown to develop prostate intraepithelial neoplasia (PIN) at a high rate (>60%) and mice with prostate specific homozygous deletion of PTEN develop invasive prostate cancer, albeit with prolonged latency of approx. 6 months. Combined inactivation of PTEN and p53 in mouse prostate elicits invasive cancer by 9 weeks of age and invariable lethality by 7 months of age. There are several PI3K pathway-directed therapies currently in Phase I clinical trials, but the underlying tumor genetic signature of patients most likely to respond to these therapies is largely unknown. To understand the significance of targeting the PI3K/mTOR pathway in advanced prostate cancer driven by PTEN +/− p53 loss, we evaluated the impact of GSK458 (a dual PI3K/mTOR inhibitor) in prostate-specific PTEN/p53 double knockout mice and prostate-specific PTEN-knockout mice. The mice were imaged by synchronized 18 FDG-PET and T2-weighted MRI, respectively, for baseline tumor metabolic and volumetric assessment prior to drug administration. GSK458 was administered at 3 mg/kg by daily oral gavage for 3 weeks with serial 18 FDG-PET and T2-weighted MRI imaging at 2 days, 1 week, 2 weeks and 3 weeks after initiation of treatment, followed by sacrifice, prostate harvest and standard hisopathologic and immunohistochemical staining. GSK458 treatment of PTEN/p53-deficient and PTEN-deficient mice results in target inhibition, based on pharmacodynamic assessment by 18 FDG-PET uptake. MRI and histopathologic analysis demonstrate that there is a significant reduction, but not complete regression of tumor burden in both intraepithelial and poorly differentiated atypical components within stroma and partial stromal collapse following 3 weeks of GSK458 treatment. These data highlight the feasibility of monitoring dual pharmacodynamics/antitumor effects of PI3K-directed therapies using 18 FDG-PET/MRI imaging and underscore the utility of genetically engineered mouse models to predict response to targeted therapies in genetically stratified human clinical trials. The evaluation of PI3K-isoform specific inhibitors and the design of rational combinations to overcome de novo and acquired resistance mechanisms to PI3K-directed therapies are currently being explored in multiple genetically engineered mouse model systems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-418. doi:10.1158/1538-7445.AM2011-LB-418