Abstract: Obesity is a major factor central to the development of insulin resistance and type 2 diabetes. The identification and characterization of genes involved in regulation of adiposity, insulin sensitivity, and glucose uptake are key to the design and development of new drug therapies for this disease. In this study, we show that the polarity kinase Par-1b/MARK2 is required for regulating glucose metabolism in vivo . Mice null for Par-1b were lean, insulin hypersensitive, resistant to high-fat diet-induced weight gain, and hypermetabolic. 18 F-FDG microPET and hyperinsulinemic–euglycemic clamp analyses demonstrated increased glucose uptake into white and brown adipose tissue, but not into skeletal muscle of Par-1b null mice relative to wild-type controls. Taken together, these data indicate that Par-1b is a regulator of glucose metabolism and adiposity in the whole animal and may be a valuable drug target for the treatment of both type 2 diabetes and obesity.

Abstract: Glutaminase (GLS1/2) catalyzes the conversion of L-glutamine to L-glutamate and ammonia. A splice variant of GLS1 (GAC) is elevated in certain cancers and is specifically inhibited by bis-2-(5-phenylacetimido-1,2,4, thiadiazol-2-yl)ethyl sulfide (BPTES). We describe here the first full length crystal structure of GAC both in the presence and absence of BPTES molecules. Two BPTES molecules bind at a interface region of the GAC tetramer in a manner which appears to lock the GAC tetramer into a non-productive conformation. The importance of these loops with regards to overall enzymatic activity of the tetramer was revealed by a series of GAC point mutants designed to create a BPTES resistant GAC. An unintended but nonetheless interesting transfer of phosphate activation activity was observed upon inserting key residues from GLS-2 into GLS-1. 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 3237. doi:1538-7445.AM2012-3237

Abstract: Genetic and epigenetic alterations in the Wnt signaling pathway leading to constitutive activation of the driver oncogene Β-catenin occur in at least 20% of all human cancers. We have developed conformationally hyperstabilized α-helical peptides (Helicons) that bind directly to Β-catenin with picomolar affinity and block its interaction with TCF transcription factors. We describe here the characterization of anti-tumor efficacy, pharmacodynamic biomarkers and mechanism of action using multiple in vivo patient-derived xenograft (PDX) models treated with Helicons. Helicon treatment leads to dose-dependent anti-tumor effects and durable regressions in PDX models from multiple indications with Wnt pathway activating mutations. Anti-tumor responses are seen in the presence of additional driver mutations, including KRAS and PIK3CA. RNA-sequencing and Gene Set Enrichment Analysis confirm Helicon treatment inhibits both Wnt/Β-catenin and MYC-regulated gene sets. Inhibiting Β-catenin-TCF interaction with Helicons represents a first-in-class therapeutic approach for the treatment of cancers resulting from aberrant transcriptional signaling via Β-catenin. Citation Format: YaGuang Si, Minjung Choi, Xinwei Han, Brian White, Ziyang Wu, Erica Visness, Pieter Beerepoot, Elizabeth Jaensch, Jessica Ramirez, Charles Ponthier, Xiaogang Han, Paula Ortet, Peicheng Du, Sorabh Agarwal, Mirek Lech, Aaron Fulgham, Sarah Cappucci, Zhi Li, John McGee, Lihua Yu, Martin Tremblay, Keith Orford, Gregory Verdine, Jonathan Hurov. Anti-tumor activity of Helicon inhibitors of Β-catenin-TCF interaction in patient-derived xenograft models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4972.

Abstract: LKB1 deficiency is found in approximately 10% of non-small cell lung cancer harboring Kras mutation. Loss of LKB1 has been associated with increased metastatic rates and decreased survival in patients. Currently, no effective therapy has been developed against this subtype of lung cancer. LKB1 can regulate multiple cellular activities. One of the most well-studied functions is its regulation on cell metabolism through AMPK signaling pathway. How LKB1 loss globally impacts NSCLC metabolism has not been well understood yet. Using isotope-labeled substrate tracing approach, we found an increase of specially-labeled intermediates in pentose phosphate pathway (PPP) that may suggest upregulation of PPP activity. In addition, we also performed an unbiased drug screen that showed higher sensitivity to an mTOR inhibitor in Kras and LKB1 double mutant lung cancer cells relative to cancer cells under other genetic background. These data suggest that loss of LKB1 may upregulate biosynthetic reactions in Kras mutant NSCLC and render these cells sensitive to inhibitors in biosynthesis. These findings may provide new insights in developing therapeutic agents targeting KrasMT/LKB1MT NSCLC. Citation Format: Tingyu Liu, Erin Sennott, Zhengjie Zhong, Mya Steadman, Kelly Marsh, Jonathan Hurov, Cyril Benes, Jeffrey A. Engelman. LKB1 deficiency is associated with a unique metabolic signature in Kras mutant non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 7.

Abstract: The phosphoinositide family of lipids includes seven derivatives of phosphatidylinositol (PI) that are formed through the phosphorylation of the 3-, 4-, and 5-positions on the inositol ring. Phosphoinositides have distinct biological roles and regulate many cellular processes, including proliferation, survival, glucose uptake, and migration. Phosphoinositide kinases, phosphatases, and phospholipases spatially and temporally regulate the generation of the different phosphoinositide species, which localize to different subcellular compartments. Phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) is synthesized by phosphoinositide 3-kinase (PI3K) and serves as the plasma membrane docking site for a subset of proteins that have pleckstrin-homology (PH) domains that bind this lipid, including the serine/threonine protein kinase AKT (also known as protein kinase B or PKB). AKT is a proto-oncogene that has critical regulatory roles in insulin signaling and cancer progression. Phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) is the major substrate for Class I PI3Ks and has a significant role itself in mediating the localization of proteins to the plasma membrane and in nucleating cortical actin polymerization (1). Until 1997 it was thought that PI-4,5-P2 was produced exclusively by phosphorylation of phosphatidylinositol-4-phosphate (PI-4-P) at the 5 position of the inositol ring, a reaction catalyzed by the Type 1 PI-4-P 5-kinases (encoded by the genes PIP5K1A, B and C). Unexpectedly, a second highly-related family of PIP kinases (called Type 2) was found to produce PI-4,5-P2 by phosphorylating the 4 position of phosphatidylinositol-5-phosphate (PI-5-P), a lipid that had been previously overlooked due to its co-migration with the much more abundant PI-4-P (2-3). The Type 2 PIP kinases are not present in yeast but are conserved in higher eukaryotes from worms and flies to mammals. Humans and mice have three distinct genes, PIP4K2A, B and C encoding enzymes called PI5P4Kα, β, and γ, respectively. The bulk of PI-4,5-P2 in most tissues is almost certainly derived from the Type 1 PIP5Ks, yet recent quantitative proteomic studies on cell lines have revealed a higher abundance of PI5P4Ks than PI4P5Ks (4). This high abundance of the Type 2 enzymes may, in part, explain why the substrate, PI-5-P is present at very low levels. While the Type 1 PIP kinases generate PI-4,5-P2 at the plasma membrane, the Type 2 kinases are located at internal membranes, including the ER, Golgi and nucleus and probably generate PI-4,5-P2 at those locations (5-8). The vast majority of PI-4,5-P2 is located at the plasma membrane and it is not clear whether the critical function of the Type 2 PIP kinases is to generate PI-4,5-P2 at intracellular sites or to maintain low levels of PI-5-P (or both). In a previous study we generated mice in which one of the Type 2 PIP kinase genes (PIP4K2B) was deleted in the germline. These mice were viable, exhibited enhanced insulin sensitivity and enhanced insulin-dependent activation of AKT in skeletal muscle (9). Paradoxically, despite increased AKT activation the mice were smaller and had decreased adiposity on a high fat diet. Cell based assays revealed that PI5P4Kβ (encoded by PIP4K2B) becomes phosphorylated by p38 at Ser326 in response to cellular stresses, such as UV and H2O2, and that this causes inhibition of the PI5P 4-kinase activity and results in increased cellular PI-5-P levels (10). These studies suggest that the Type 2 PIP kinases mediate cellular stress responses downstream of p38 (presumably by altering the PI-5-P/PI-4,5-P2 ratio at intracellular locations) and that under conditions of low stress, these enzymes suppress the PI3K/AKT signaling pathway. It should be pointed out that the Type 2 PIP kinases are unlikely to supply PI-4,5-P2 as a substrate for PI3K since activation of AKT correlates with loss of PI5P4K activity rather than gain. In this study we have interrogated the potential role of Type 2 PIP kinases in cancers. We found high levels of either PI5P4Kα or PI5P4Kβ enzymes or both in a number of breast cancer cell lines, and more importantly, found amplification of the PIP4K2B gene and high levels of both the PI5P4Kα and PI5P4Kβ proteins in a subset of human breast tumors. We found that knocking down the levels of both PI5P4Kα and PI5P4Kβ in a TP53 deficient breast cancer cell line blocked growth on plastic and in xenografts. This impaired growth correlated with impaired glucose metabolism and enhanced levels of reactive oxygen species (ROS) leading to senescence. The impaired glucose metabolism, despite activation of the PI3K-AKT pathway (which typically enhances glucose metabolism) was paradoxical. The results indicate that PI3K activation is not driving the ROS production, but may be an inadequate feedback attempt to restore glucose uptake and metabolism. To assess the role of Type 2 PIP kinases in tumor formation, we generated mice with germline deletions of PIP4K2A and PIP4K2B and crossed these with TP53-/- mice and evaluated tumor formation in all the viable genotypes. We found that mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly, mice with the genotype PIP4K2A-/-, PIP4KB+/-, TP53-/- were viable and had a dramatic reduction in tumor formation compared to siblings that were TP53-/- and wild type for PIP4K2B and/or PIP4K2A genes. The decreased tumor incidence in the background of PIP4K2A-/-, PIP4K2B+/-, TP53-/- compared to TP53-/- alone is particularly interesting in respect to Li Fraumeni Syndrome (germline TP53 mutations). Our results indicate that expression of PI5P4Kα and/or β is critical for the growth of tumors with TP53 mutations or deletions. Thus, co-amplification of PIP4K2B with ERBB2 might explain why breast cancers in patients with Li Fraumeni syndrome show ERBB2 amplifications (HER2-positive) in over 83% of cases as opposed to 16% of age-matched patients with wild type TP53 (11). The results that we present here suggest that PI5P4Kα and β play a critical role in mediating changes in metabolism in response to stress, and in particular ROS stress that occurs in the absence of p53. Germ-line deletion of either PIP4K2A or PIP4K2B alone resulted in mice with normal lifespans, and germ-line deletion of both PIP4K2A and PIP4K2B resulted in full-term embryos of normal size and appearance at birth, indicating that these genes do not play a major role in normal embryonic growth and development. Yet the PIP4K2A-/-, PIP4K2B-/- pups die shortly after birth, consistent with these genes having a role in mediating stress responses known to occur following birth. Importantly, germ-line deletion of both PIP4K2B and TP53 resulted in lethality while germ-line deletion of either gene alone resulted in Mendelian ratios of viable pups. Thus, the genetic studies suggest that TP53 and PIP4K2B have overlapping roles in mediating cellular responses to stress and that, while neither gene alone is essential, loss of both genes is not tolerated. The most exciting observation from these studies in regard to potentially new therapies for p53 mutant tumors is that germ-line deletion of both alleles of PIP4K2A and one allele of PIP4K2B in the context of TP53-/- results in a viable mouse with a dramatic reduction in tumor-dependent death compared to TP53-/- mice that are wild type for PIP4K2A and B. These results (and studies of the PIP4K2A-/-, PIP4K2B+/- or PIP4K2A+/-, PIP4K2B-/- mice in the context of wild type TP53) indicate that normal tissues tolerate well the loss of three out of four alleles of the PIP4K2A and PIP4K2B genes, but that tumors are not viable in this context. PI5P4Kα and β are kinases and pharmaceutical companies have shown that it is possible to develop highly specific inhibitors of both protein kinases and lipid kinases. The synthetic lethality that we observe between TP53 loss and loss of these kinases indicates that drugs that target either the enzyme PI5P4Kβ alone or that target both PI5P4Kα and β are likely to be well-tolerated and very effective on tumors that have loss of function mutations or deletions of TP53. Our observations with BT474 cells suggest that HER2 positive tumors that have amplifications of PIP4K2B and mutations in TP53 may be particularly sensitive to PI5P4Kα,β inhibitors. The ERBB2 (Her2) amplicon on chromosome 17 is variable in size and can contain a number of cancer-related genes in addition to the ERBB2 locus. Clinically, patients who have tumors with small amplicons confined to the ERBB2 locus have the greatest benefit from ERBB2-directed therapies such as Trastuzumab, while tumors with wider ERBB2 amplicons have poor responses, suggesting co-amplification of genes that contribute to Trastuzumab resistance (11). PIP4K2B, which is located in a chromosomal region (17q12) close to ERBB2, may be a candidate for an adjacent co-amplified gene that confers Trastuzumab resistance, and, conversely, concomitant inhibition of ERBB2 and PIP4K2B could be a highly effective treatment option for ERBB2 (Her2) positive tumors that are p53-mutant and PIP4K2B-amplified. References 1. Cantley, L.C. The phosphoinositide 3-kinase pathway. Science 2002;296:1655-1657. 2. Rameh, L.E., and Cantley, L.C. The role of phosphoinositide 3-kinase lipid products in cell function. J Biol Chem 1999;274:8347-8350. 3. Rameh, L.E., Tolias, K.F., Duckworth, B.C., and Cantley, L.C. A new pathway for synthesis of phosphatidylinositol-4,5-bisphosphate. Nature 1997;390:192-196. 4. Nagaraj, N., Wisniewski, J.R., Geiger, T., Cox, J., Kircher, M., Kelso, J., Paabo, S., and Mann, M. Deep proteome and transcriptome mapping of a human cancer cell line. Mol Syst Biol 2011;7: 548. 5. Fruman, D.A., Meyers, R.E., and Cantley, L.C. Phosphoinositide kinases. Annu Rev Biochem 1998;67:481-507. 6. Sarkes, D., and Rameh, L.E. A novel HPLC-based approach makes possible the spatial characterization of cellular PtdIns5P and other phosphoinositides. Biochem J 2010;428:375-384. 7. Schaletzky, J., Dove, S.K., Short, B., Lorenzo, O., Clague, M.J., and Barr, F.A. Phosphatidylinositol-5-phosphate activation and conserved substrate specificity of the myotubularin phosphatidylinositol 3-phosphatases. Curr Biol 2003;13:504-509. 8. Walker, D.M., Urbe, S., Dove, S.K., Tenza, D., Raposo, G., and Clague, M.J. Characterization of MTMR3. an inositol lipid 3-phosphatase with novel substrate specificity. Curr Biol 2001;11:1600-1605. 9. Lamia, K.A., Peroni, O.D., Kim, Y.B., Rameh, L.E., Kahn, B.B., and Cantley, L.C. Increased insulin sensitivity and reduced adiposity in phosphatidylinositol 5-phosphate 4-kinase beta-/- mice. Mol Cell Biol 2004;24:5080-5087. 10. Jones, D.R., Bultsma, Y., Keune, W.J., Halstead, J.R., Elouarrat, D., Mohammed, S., Heck, A.J., D'Santos, C.S., and Divecha, N. Nuclear PtdIns5P as a transducer of stress signaling: an in vivo role for PIP4Kbeta. Mol Cell 2006;23:685-695. 11. Morrison, L.E., Jewell, S.S., Usha, L., Blondin, B.A., Rao, R.D., Tabesh, B., Kemper, M., Batus, M., and Coon, J.S. Effects of ERBB2 amplicon size and genomic alterations of chromosomes 1, 3, and 10 on patient response to trastuzumab in metastatic breast cancer. Genes Chromosomes Cancer 2007;46:397-405. Citation Format: Brooke M. Emerling, Jonathan B. Hurov, George Poulogiannis, Rayman Choo-Wing, Gerburg M. Wulf, Hye-Seok Shim, Katja A. Lamia, Lucia E. Rameh, Xin Yuan, Andrea Bullock, Gina M. DeNicola, Jiaxi Song, Sabina Signoretti, Lewis C. Cantley. Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53 null tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr NG05. doi:10.1158/1538-7445.AM2014-NG05

Abstract: Transcriptomics and sequencing analyses of tumors from patients provide invaluable information about the cells present in the tumor microenvironment (TME); however, nerves, whose cell bodies are absent from most peripheral TMEs, have eluded such approaches. Recent emerging data highlight the functional importance of innervation in the TME and its contribution to tumor progression, metastasis, and treatment resistance. The Exoneural Platform developed at Cygnal Therapeutics allows for investigation of complex biology in the context of multi-cell culture conditions in vitro and in vivo. As part of this platform, we have developed tools for rapid and selective manipulation of different cell types with the ability to monitor multiple cell specific read-outs. Here, we used a combination of approaches including RNA-seq, bioinformatics, immunohistochemistry, and microscopy to investigate the role of synaptic function in cancer biology. Transcriptomics analyses on co-cultures revealed a direct interaction between nerves and cancer cells based on cell type specific gene expression and ligand - receptor interaction assessment. Both neurons and cancer cells showed a profound shift in their gene expression profile when cultured together. This differential gene expression was reversed if neurons were ablated, pointing to the reversibility of this change and, potentially, the disease state itself. Examination for presence and function of synaptic proteins showed that synaptic proteins, such as PSD95 and synapsin1, were expressed in co-cultures, and in many cases, a co-localization of pre - and post - synaptic markers was observed. Selective stimulation of dorsal ganglion root (DRG) neurons, co-cultured with cancer cells, resulted in a robust and acute increase of cytosolic calcium in cancer cells. Bioinformatic image analysis confirmed that this calcium influx in cancer cells directly correlated with proximity and density of neurites to cancer cells. A calcium response was not observed if DRGs and cancer cells were cultured in two separate chambers between which media can freely travel, confirming that close proximity is required for this interaction. Pharmacological and genetic knock down of key synaptic proteins resulted in changes in cancer cell calcium influx and affected proliferation and gene expression. Bioinformatics analyses, based on public and proprietary data, have identified the synaptic pathway as a major contributing node that influences cancer cell biology in the context of several cancer types. These data collectively point to the importance of exoneural biology, and more specifically, synaptic biology in cancer. More importantly, these results are likely to suggest new oncology targets that have not been identified or pursued previously. Citation Format: Monica Thanawala, Chih-Chieh Wang, Jesse G. Turner, Kai-Chih Huang, Lexiang Ji, Alison Miller, Alexandria Fink, Shan Lou, Alexandra B. Lantermann, Hongyue Dai, John A. Wagner, Grazia Piizi, Jonathan B. Hurov, Pearl Huang, Amir M. Sadaghiani. Neural communication to peripheral tumors regulates cancer cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1450.

Abstract: Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA-damage response pathways.

Abstract: To produce blood platelets, the megakaryocyte (MK) cytoplasm elaborates proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. Invaginated demarcation membranes (DMS) are thought to be the source for the proplatelet and platelet membranes, however, they have THUS far BEEN INSUFFICIENTLY characterized. We first used a mouse model where the cDNA encoding enhanced yellow fluorescence protein (EYFP) with a C-terminally introduced myristoyl acceptor site has been introduced into the GPIIb locus. Heterozygous knock-in mice reveal yellow fluroescent MKs with an internal staining pattern that resembles the reticiulated pattern of the DMS as found in micrographs. Proplatelet-forming MKs reveal contiguous membrane connection between the internally stained membranes and the outlines of the proplatelet shaft resulting in production of fluorescent platelets. We next sought to characterize the internal membranes biochemically and retrovirally infected MKs to express the green fluorescence protein (EGFP) tagged with the pleckstrin homology domain of phospholipase Cδ1 (PLCδ1) which binds with high specificity to phosphatidylinositol(4,5)P2 (PIP2). Young MKs stain the cell periphery as described for most other cell types. Mature MKs, however, stain the internal membranes, whereas the plasma membrane becomes PIP2-negative as shown by co-staining with CD41. Proplatelet membranes emanate from these internal PIP2-positive membranes, proving that the DMS is indeed the membrane reservoir during platelet biogenesis. Appearance of PI-4,5-P2 in the DMS occurs in proximity to PI-5-P-4-kinaseα (PI4Kα), a protein highly expressed in MKs and platelets, as shown by overexpressing EGFP-tagged kinase in primary MKs. In addition, shRNA-mediated loss of PIP4Kα or depletion of its presumptive substrate block DMS development and expansion of MK size. Thus, PI-4,5-P2 is a marker and essential component of internal membranes and is most likely introduced about the non-canonical pathway using PI5P as the substrate. PI-4,5-P2 promotes actin polymerization by activating small GTPases from the Rac/Rho superfamily as well as Wiskott-Aldrich Syndrome (WASp) family proteins. Indeed, PIP2 is associated with filamentous actin when MKs are co-stained with phalloidin. Expression of a dominant-negative N-WASp C-terminal fragment (CA-domain) that inactivats all WASp/WAVE family members leads to Arp3 binding without assembling the complete Arp2/3 complex, thus inhibiting actin filament nucleation. F-Actin staining in the infected MKs reveals a pattern similar to that of MKs treated with pharmacologic dosage of actin polymerization-antagonists like cytochalasin D, which disrupts actin filaments and inhibits proplatelet formation when administered early in MK culture. Dominant-negative WASp impairs proplatelet elaboration similarly, acting at a step past expansion of the cell volume. These observations implicate a signaling pathway wherein PI-4,5-P2 facilitates DMS development and suggests a pathway that links a DMS lipid marker with local assembly of actin fibers as a requirement for platelet biogenesis.