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Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K–PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

Supplementary Table 5. Patient B Single and buparlisib Combination High Throughput Drug Screen.

Supplementary Figures 1 - 6

Supplementary Table 2. Drug Library Concentrations.

Supplementary Table 3. Patient A single and duparlisib combination high throughout drug screen.

Supplementary Table 1. Genomic analysis of 15 patients.

Supplementary Table Legends

Supplementary Table 7. Patient D SINGLE and afatinib COMBINATION High Throughput Drug Screen.

Supplementary Table 4. Patient A Single and vorinostat COMBINATION High Throughput Drug Screen.

Supplementary Information, Methods, Table 1, Figures 1-7 from 3-Phosphoinositide–Dependent Kinase 1 Potentiates Upstream Lesions on the Phosphatidylinositol 3-Kinase Pathway in Breast Carcinoma

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. [Cancer Res 2009;69(15):6299–306]

Obesity, defined as a body mass index (BMI) ≥ 30, is an established risk factor for breast cancer among women in the general population after menopause. Whether elevated BMI is a risk factor for women with a germline mutation in BRCA1 or BRCA2 is less clear because of inconsistent findings from epidemiological studies and a lack of mechanistic studies in this population. Here, we show that DNA damage in normal breast epithelia of women carrying a BRCA mutation is positively correlated with BMI and with biomarkers of metabolic dysfunction. In addition, RNA sequencing showed obesity-associated alterations to the breast adipose microenvironment of BRCA mutation carriers, including activation of estrogen biosynthesis, which affected neighboring breast epithelial cells. In breast tissue explants cultured from women carrying a BRCA mutation, we found that blockade of estrogen biosynthesis or estrogen receptor activity decreased DNA damage. Additional obesity-associated factors, including leptin and insulin, increased DNA damage in human BRCA heterozygous epithelial cells, and inhibiting the signaling of these factors with a leptin-neutralizing antibody or PI3K inhibitor, respectively, decreased DNA damage. Furthermore, we show that increased adiposity was associated with mammary gland DNA damage and increased penetrance of mammary tumors in Brca1 +/− mice. Overall, our results provide mechanistic evidence in support of a link between elevated BMI and breast cancer development in BRCA mutation carriers. This suggests that maintaining a lower body weight or pharmacologically targeting estrogen or metabolic dysfunction may reduce the risk of breast cancer in this population.

The phosphoinositide 3-kinase (PI3K) pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers. A comprehensive elucidation of how this signaling pathway controls transcriptional and cotranscriptional processes could provide new insights into the key functions of PI3K signaling in cancer. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced isoforms linked to proliferation, metabolism, and splicing in PIK3CA-mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from patients with PIK3CA-mutated breast cancer undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer.Transcriptomic analysis reveals a key role for the PI3K pathway in regulating RNA splicing, uncovering new mechanisms by which PI3K regulates proliferation and metabolism in breast cancer. See related commentary by Claridge and Hopkins, p. 2216.

Obesity is an established risk factor for breast cancer among women in the general population after menopause. Whether elevated bodyweight is a risk factor for women with a germline mutation in BRCA1 or BRCA2 is less clear due to inconsistent findings from epidemiological studies and lack of mechanistic studies in this population. Here, we show that DNA damage in normal breast epithelium of BRCA mutation carriers is positively correlated with body mass index and with biomarkers of metabolic dysfunction. Additionally, RNA-sequencing reveals significant obesity-associated alterations to the breast adipose microenvironment of BRCA mutation carriers, including activation of estrogen biosynthesis, which impacts neighboring breast epithelial cells. We found that blockade of estrogen biosynthesis or estrogen receptor activity decreases DNA damage, whereas treatment with leptin or insulin increases DNA damage in BRCA heterozygous epithelial cells. Furthermore, we show that increased adiposity is associated with mammary gland DNA damage and increased penetrance of mammary tumors in Brca1+/- mice. Overall, our results provide mechanistic evidence in support of a link between bodyweight and breast cancer development in BRCA mutation carriers and suggests that maintaining a healthy bodyweight or pharmacologically targeting estrogen or metabolic dysfunction may reduce the risk of breast cancer in this population.One Sentence SummaryElevated bodyweight is positively associated with DNA damage in breast epithelium of BRCA mutation carriers

SUMMARYThe phosphoinositide 3-kinase, p110α, is an essential mediator of insulin signaling and glucose homeostasis. We systematically interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate and completely inhibit its activity. This inhibitory state corresponds to a conformational change of a membrane binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases, MST1/2, using forskolin or epinephrine is associated with phosphorylation and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules. Our study reveals a novel inhibitory pathway of PI3K signaling and a previously unappreciated link between epinephrine and insulin signaling.

The United States has been at war in Afghanistan for nearly twenty years, with arguably no discernible end in sight. Yet there has been no public accounting of the costs of the on-going conflict, e...

Fumarate is an oncometabolite. However, the mechanism underlying fumarate-exerted tumorigenesis remains unclear. Here, utilizing human type2 papillary renal cell carcinoma (PRCC2) as a model, we show that fumarate accumulates in cells deficient in fumarate hydratase (FH) and inhibits PTEN to activate PI3K/AKT signaling. Mechanistically, fumarate directly reacts with PTEN at cysteine 211 (C211) to form S-(2-succino)-cysteine. Succinated C211 occludes tethering of PTEN with the cellular membrane, thereby diminishing its inhibitory effect on the PI3K/AKT pathway. Functionally, re-expressing wild-type FH or PTEN C211S phenocopies an AKT inhibitor in suppressing tumor growth and sensitizing PRCC2 to sunitinib. Analysis of clinical specimens indicates that PTEN C211 succination levels are positively correlated with AKT activation in PRCC2. Collectively, these findings elucidate a non-metabolic, oncogenic role of fumarate in PRCC2 via direct post-translational modification of PTEN and further reveal potential stratification strategies for patients with FH loss by combinatorial AKTi and sunitinib therapy.

Objective Diagnosis of patients with occipital headache can be challenging, as both primary and secondary causes must be considered. Our study assessed how often migraine is screened for, diagnosed, and treated in patients receiving greater occipital nerve blocks (GONBs) in a pain clinic. Design Institutional review board–approved, retrospective observational study. Setting Academic multidisciplinary pain clinic. Subjects One hundred forty-three consecutive patients who received GONBs. Results About 75% of patients had been evaluated by neurologists and about 25% by non-neurologist pain specialists only, and 62.2% of patients had photophobia, phonophobia, and nausea assessed. Compared with patients who had been evaluated by non-neurologists, patients who had been evaluated by a neurologist were more likely to have photophobia, phonophobia, and nausea assessed (75.9% vs 20.0%, odds ratio [OR] 12.6, 95% confidence interval [CI] 4.90 to 32.2); more likely to be diagnosed with migraine (48.1% vs 14.3%, OR 5.6, 95% CI 2.0 to 15); less likely to be diagnosed with occipital neuralgia (39.8% vs 65.7%, OR 0.3, 95% CI 0.2 to 0.8); and equally likely to be diagnosed with cervicogenic headache (21.3% vs 25.7%, OR 0.8, 95% CI 0.3 to 1.9). Among patients diagnosed with migraine, 82.5% received acute migraine treatment, 89.5% received preventive migraine treatment, and 52.6% were documented as receiving migraine lifestyle counseling. Conclusions Of the patients in this study who had occipital headache and received GONBs, 62.2% were assessed for migraine, and most received appropriate acute, preventive, and lifestyle treatments when diagnosed. Patients seen by neurologists were significantly more likely to be screened for and diagnosed with migraine than were those evaluated by non-neurologist pain medicine specialists only. All clinicians should remain vigilant for migraine in patients with occipital headache.

Over the last two decades, cancer researchers have taken the promise offered by the Human Genome Project and have expanded its capacity to use sequencing to identify the genomic alterations that give rise to and sustain individual tumors. This expansion has allowed researchers to identify and target highly recurrent alterations in specific cancer contexts, such as EGFR mutations in non-small cell lung cancer (Lynch et al, N Engl J Med 350:2129-2139, 2004; Sharifnia et al., Proc Natl Acad Sci U S A 111:18661-18666, 2014), BCR-ABL translocations in chronic myeloid leukemia (Deininger, Pharmacol Rev 55:401-423. https://doi.org/10.1124/pr.55.3.4 , 2003; Druker et al, N Engl J Med 344. 1038-1042, 2001; Druker et al, N Engl J Med 344:1031-1037. https://doi.org/10.1056/NEJM200104053441401 , 2001), or HER2 amplifications in breast cancer (Slamon et al, N Engl J Med 344:783-792. https://doi.org/10.1056/NEJM200103153441101 , 2001; Solca et al, Beyond trastuzumab: second-generation targeted therapies for HER-2-positive breast cancer. In: Sibilia M, Zielinski CC, Bartsch R, Grunt TW (eds) Drugs for HER-2-positive breast cancer. Springer, Basel, pp 91-107, 2011). Despite these advances in our capacity to identify the genetic alterations that drive tumor initiation, survival, and proliferation, our ability to target these alterations to provide effective treatment options for patients in need, particularly those with rare or advanced cancers, remains limited (Gould et al, Nat Med 21:431-439. https://doi.org/10.1038/nm.3853 , 2015). Patient-derived models of cancer offer one potential mechanism to overcome this barrier between the bench and bedside. Through the development and testing of patient-derived models of cancer, functional genomics efforts can identify tumor-specific drug sensitivities and thereby provide a connection between tumor genetics and effective therapeutics for patients in need of treatment options.Recognizing that cancer is a multifaceted set of disease states, the development of personalized models of cancer that can be used to compare treatment options, identify tumor-specific vulnerabilities, and guide clinical decision-making has tremendous potential for improving patient outcomes. This chapter will describe a representative set of patient-derived models of cancer, reviewing each of their strengths and weaknesses and highlighting how selecting a model to suit a specific question or context is critical. Each model comes with a unique set of pros and cons, making them more or less appropriate for each specific research or clinical question. As each model can be leveraged to gain new insights into cancer biology, the key to their deployment is to identify the most appropriate model for a specific context, while carefully considering the strengths and limitations of the selected model. When used appropriately, patient-derived models may prove to be the missing link needed to bring the promise of personalized oncology to fruition in the clinic.

Cullin-RING E3 ubiquitin ligase 4 (CRL4) plays an essential role in cell cycle progression. Recent efforts using high throughput screening and follow up hit-to-lead studies have led to identification of small molecules 33-11 and KH-4-43 that inhibit E3 CRL4’s core ligase complex and exhibit anticancer potential. This review provides: 1) an updated perspective of E3 CRL4, including structural organization, major substrate targets and role in cancer; 2) a discussion of the challenges and strategies for finding the CRL inhibitor; and 3) a summary of the properties of the identified CRL4 inhibitors as well as a perspective on their potential utility to probe CRL4 biology and act as therapeutic agents.

Tumors exhibit widespread enhancer landscape reprogramming compared to normal tissue. The etiology is believed to be largely cell-intrinsic in non-hormonal cancers, attributed to such genomic alterations as focal amplification of non-coding regions, aberrant activation of transcription factors, and non-coding mutations creating de novo transcription factor binding sites. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon epithelia, we find divergent epigenetic landscapes between primary CRC tumors and CRC cell lines. We identify a unique super-enhancer signature largely absent in cell culture. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over patient-matched normal epithelium suggesting novel insight into the etiology of enhancer reprogramming in CRC and its downstream relevance to tumor biology. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating its etiology via local tumor microenvironment acquisition. Building on its known role in glucose uptake via SGLT receptors, we demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway using polar metabolomic profiling. We show this activation enables efficient growth under oxidative conditions both in vitro and in vivo, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the biologic significance of epigenomic profiling on patient-matched primary specimens and identify this microenvironment-acquired super-enhancer as an oncogenic driver in the setting of the inflamed tumor.

The uncontrolled cellular growth that characterizes tumor formation requires a constant delivery of nutrients. Since the 1970s, researchers have wondered if the supply of nutrients from the diet could impact tumor development. Numerous studies have assessed the impact of dietary components, specifically sugar and fat, to increased cancer risk. For the most part, data from these trials have been inconclusive; however, this does not indicate that dietary factors do not contribute to cancer progression. Rather, the dietary contribution may be dependent on tumor, patient, and context, making it difficult to detect in the setting of large trials. In this review, we combine data from prospective cohort trials with mechanistic studies in mice to argue that fat and sugar can play a role in tumorigenesis and disease progression. We find that certain tumors may respond directly to dietary sugar (colorectal and endometrial cancers) and fat (prostate cancer) or indirectly to the obese state (breast cancer).

In this era of precision medicine, numerous workflows for the targeting of high-recurrent mutations in common tumor types have been developed, leaving patients with rare diseases with few options. Here, we implement a functional precision oncology approach utilizing comprehensive genomic profiling in combination with high-throughput drug screening, to identify tumor-specific drug sensitivities for patients with rare tumor types such as myxofibrosarcoma. From a patient with a high-grade myxofibrosarcoma, who was enrolled in the Englander Institute for Precision Medicine (EIPM) program, we established patient-derived 3D sarco-spheres and xenograft models for functional testing. In the absence of a large cohort of clinically similar cases, high-throughput drug screening was performed on the patient-derived cells, and compared with two other myxofibrosarcoma lines and a benign fibroblast line to functionally identify tumor-specific drug sensitivities. The addition of functional drug sensitivity testing to complement genomic profiling identified multiple therapeutic options that were further validated in patient derived xenograft models. Genomic analyses detected the frequently known codeletion of the tumor suppressors CDKN2A/B together with the methylthioadenosine phosphorylase (MTAP) and a TP53 E286fs*50 mutation. High-throughput drug screening demonstrated tumor-specific sensitivity to compounds targeting the cell cycle. Based on genomic analysis and high-throughput drug screening, we show that targeting the cell cycle in these tumors is a powerful approach. Implications: This study demonstrates the potential of functional testing to aid clinical decision making for patients with rare or molecularly complex malignancies when combined with comprehensive genomic profiling.

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis

Significance Cullin-RING E3 ubiquitin ligases (CRLs) direct protein degradation to impact a myriad of physiological and pathological processes including cancer. This work reports the small-molecule compounds 33-11 and KH-4-43 as inhibitors of E3 CRL4 with anticancer potential. These compounds have provided an opportunity for developing tool compounds to address mechanistic and phenotypic questions about CRL4 in biochemical, cell-based, and animal studies. The results of correlation studies between cullin4 protein level and the compounds’ cytotoxic response as well as cullin4 depletion experiments suggest a role for low E3 abundance in sensitizing tumor cells for apoptosis. Collectively, the 33-11/KH-4-43–based CRL4 inhibitors may provide new exploitable therapeutic opportunities to target a subset of tumor lines characterized by low CUL4 expression., Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4’s core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4’s substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds’ cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4–expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.

Obesity increases the risk and worsens the prognosis for breast cancer due, in part, to altered adipose stromal cell (ASC) behavior. Whether ASCs from obese individuals increase migration of breast cancer cells relative to their lean counterparts, however, remains unclear. To test this connection, multicellular spheroids composed of MCF10A-derived tumor cell lines of varying malignant potential and lean or obese ASCs were embedded into collagen scaffolds mimicking the elastic moduli of interstitial breast adipose tissue. Confocal image analysis suggests that tumor cells alone migrate insignificantly under these conditions. However, direct cell-cell contact with either lean or obese ASCs enables them to migrate collectively, whereby obese ASCs activate tumor cell migration more effectively than their lean counterparts. Time-resolved optical coherence tomography (OCT) imaging suggests that obese ASCs facilitate tumor cell migration by mediating contraction of local collagen fibers. Matrix metalloproteinase (MMP)-dependent proteolytic activity significantly contributes to ASC-mediated tumor cell invasion and collagen deformation. However, ASC contractility is also important, as co-inhibition of both MMPs and contractility is necessary to completely abrogate ASC-mediated tumor cell migration. These findings imply that obesity-mediated changes of ASC phenotype may impact tumor cell migration and invasion with potential implications for breast cancer malignancy in obese patients.

Cancer is driven by incremental changes that accumulate, eventually leading to oncogenic transformation. Although genetic alterations dominate the way cancer biologists think about oncogenesis, growing evidence suggests that systemic factors (for example, insulin, oestrogen and inflammatory cytokines) and their intracellular pathways activate oncogenic signals and contribute to targetable phenotypes. Systemic factors can have a critical role in both tumour initiation and therapeutic responses as increasingly targeted and personalized therapeutic regimens are used to treat patients with cancer. The endocrine system controls cell growth and metabolism by providing extracellular cues that integrate systemic nutrient status with cellular activities such as proliferation and survival via the production of metabolites and hormones such as insulin. When insulin binds to its receptor, it initiates a sequence of phosphorylation events that lead to activation of the catalytic activity of phosphoinositide 3-kinase (PI3K), a lipid kinase that coordinates the intake and utilization of glucose, and mTOR, a kinase downstream of PI3K that stimulates transcription and translation. When chronically activated, the PI3K pathway can drive malignant transformation. Here, we discuss the insulin-PI3K signalling cascade and emphasize its roles in normal cells (including coordinating cell metabolism and growth), highlighting the features of this network that make it ideal for co-option by cancer cells. Furthermore, we discuss how this signalling network can affect therapeutic responses and how novel metabolic-based strategies might enhance treatment efficacy for cancer.

Background75% of diagnosed bladder tumors are non-muscle-invasive (NMIBC)[1, 2]. Most require intravesical instillation of M.bovis Bacillus Calmette-Guérin (BCG). Recurrence after immunotherapy occurs in ~50% patients. Development of treatments for BCG-resistant disease has lagged partly because few studies have attempted to understand the relationship between timing of tumor recurrence, reasoning for the recurrence, and the state of immune system at the time of recurrence.Immune exhaustion is observed following microbial infections, cancers and chronic inflammation [3–5]. Natural Killer (NK) cells are among the earliest responders[6–8] and undergo a similar program of exhaustion as T cells[9]. HLA-E strongly inhibits NKG2A-expressing NK and CD8+T cells and is commonly upregulated on tumors[10]. We evaluated the potential restorative capacity of NKG2A and PD-L1-blockade for reinvigorating NK and CD8+T cell antitumor functions in in BCG-resistant bladder cancer.Methods mRNA analysis of 2,892 genes was performed on tumor tissue of NMIBC patients before and after BCG therapy (n=35). Immunostaining (serial-IHC,immunofluorescence,imaging-mass cytometry) was performed on consecutive tissue sections. Single-cell-RNA-sequencing (scRNAseq) was performed on fresh bladder tumors (NMIBC,n=4; MIBC,n=9). OLink Proteomics (”Inflammation” panel) was performed longitudinally on plasma/urine from a prospective cohort of NMIBC patients. Patient tumors (n=3) were expanded as organoids and co-cultured with autologous tumor-derived NK and CD8+T cells in presence/absence of anti-PD-L1/NKG2A antibodies.ResultsWe demonstrate a robust local TME and systemic response to BCG that correlates with chronic inflammation and adaptive resistance rather than with preventing tumor recurrence. This resistance is mediated through IFN-γ-production by tumor-infiltrating NKG2A+NK and NKG2A+PD-1+CD8+T cells and results in increased HLA-E and PD-L1 on recurring tumors. Co-culture of treatment-naïve NMIBC tumors with recombinant IFN-gamma directly enhanced expression of PD-L1 and HLA-E. Longitudinal analysis of plasma before and during BCG immunotherapy revealed an inflammatory signature, including but not limited to IFN-gamma, that is maintained throughout treatment.Immunostaining and scRNAseq of NMIBC specimens revealed highly enriched infiltration by NKG2A+NK and NKG2A+CD8+T cells in HLA-EBrightPD-L1+ tumors and were spatially organized relative to tumors in a manner suggesting direct inhibition. Tumor-derived NK and CD8+T cells from BCG-resistant patients were co-cultured with autologous tumor organoids. Preliminary analyses demonstrated an improved anti-tumor response in presence of NKG2A/PD-L1-blockade.ConclusionsOur data support a model of BCG-resistance that points to a novel checkpoint axis that contributes to BCG-resistance: HLA-E/NKG2A. New insights into this axis in NMIBC and how it is altered with repeated BCG exposure will enable us to explore combination therapies (PD-L1/NKG2A-blockade) that may reduce BCG-resistance and provide durable response.ReferencesEidinger D, Morales A: Discussion paper: treatment of superficial bladder cancer in man. Ann N Y Acad Sci 1976, 277:239–240.Morales A, Eidinger D, Bruce AW: Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors. J Urol 1976, 116:180–183.Blank CU, Haining WN, Held W, Hogan PG, Kallies A, Lugli E, Lynn RC, Philip M, Rao A, Restifo NP et al: Defining ‘T cell exhaustion’. Nat Rev Immunol 2019, 19:665–674.Hashimoto M, Kamphorst AO, Im SJ, Kissick HT, Pillai RN, Ramalingam SS, Araki K, Ahmed R: CD8 T Cell Exhaustion in Chronic Infection and Cancer: Opportunities for Interventions. Annu Rev Med 2018, 69:301–318.McLane LM, Abdel-Hakeem MS, Wherry EJ: CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu Rev Immunol 2019, 37:457–495.Lanier LL: NK cell receptors. Annu Rev Immunol 1998, 16:359–393.Biron CA, Gazzinelli RT: Effects of IL-12 on immune responses to microbial infections: a key mediator in regulating disease outcome. Curr Opin Immunol 1995, 7:485–496.Welsh RM, Jr.: Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice. I. Characterization of natural killer cell induction. J Exp Med 1978, 148:163–181.da Silva IP, Gallois A, Jimenez-Baranda S, Khan S, Anderson AC, Kuchroo VK, Osman I, Bhardwaj N: Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 blockade. Cancer Immunol Res 2014, 2:410–422.van Hall T, Andre P, Horowitz A, Ruan DF, Borst L, Zerbib R, Narni-Mancinelli E, van der Burg SH, Vivier E: Monalizumab: inhibiting the novel immune checkpoint NKG2A. J Immunother Cancer 2019, 7:263.Ethics ApprovalPrimary urothelial bladder cancer tumor tissue was obtained after obtaining informed consent in the context of an Institutional Review Board (IRB)-approved genitourinary cancer clinical database and specimen collection protocol (IRB #10-1180) at the Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai (New York, NY).

Rare cancers present a distinct challenge for the identification of effective therapeutic strategies. While these tumors make up 20% of total cancer cases in the United States, they are categorically defined as occurring in less than 6 of every 100,000 people, limiting the utility of traditional methods to study, and identify treatments for these cancers. Though there have been tremendous advances in cancer therapeutics, identification of treatments for rare tumors have lagged, leaving patients with rare tumors with worse 5 year survival rates than individuals diagnosed with common cancer types. The goal of precision oncology is to identify effective therapeutic strategies for each individual patient. With high throughput sequencing it is now possible to identify the genetic events that give rise to and sustain each individual tumor in clinically relevant time frames, however our capacity to leverage this information to identify effective therapeutic options remains limited. This gap in our capacity stems from a lack of clinically relevant approaches that would allow physicians to accurately gauge the impact of multiple therapeutic strategies and relate them to clinical responses. Through the establishment of an iterative Functional Genomics Pipeline which integrates the development and analysis of high-fidelity patient derived organoid models of rare cancers with personalized drug sensitivity testing, and comprehensive genomics our work suggests that personalized modeling could have clinical utility for the identification of treatment options for individual patients with rare cancers. Citation Format: Benjamin David Hopkins, Julie-Ann Cavallo-Fleming, Sally Claridge, Lamberto De Boni, Chantal Pauli. Organoid based functional genomics approach to identify treatment options for patients with rare cancers [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 2968.

Obesity is a risk factor for cancer development and is associated with poor prognosis in multiple tumor types. The positive energy balance linked with obesity induces a variety of systemic changes including altered levels of insulin, insulin-like growth factor-1, leptin, adiponectin, steroid hormones, and cytokines. Each of these factors alters the nutritional milieu and has the potential to create an environment that favors tumor initiation and progression. Although the complete ramifications of obesity as it relates to cancer are still unclear, there is convincing evidence that reducing the magnitude of the systemic hormonal and inflammatory changes has significant clinical benefits. This review will examine the changes that occur in the obese state and review the biologic mechanisms that connect these changes to increased cancer risk. Understanding the metabolic changes that occur in obese individuals may also help to elucidate more effective treatment options for these patients when they develop cancer. Moving forward, targeted clinical trials examining the effects of behavioral modifications such as reduced carbohydrate intake, caloric restriction, structured exercise, and/or pharmacologic interventions such as the use of metformin, in obese populations may help to reduce their cancer risk.

Cancer cells can adapt to nutrient poor conditions by rewiring their metabolism and using alternate fuel sources. Identifying these adaptive metabolic pathways may provide novel targets for cancer therapy. Here, we identify a subset of non-small cell lung cancer (NSCLC) cell lines that survive in the absence of glucose by internalizing and metabolizing extracellular protein via macropinocytosis. Macropinocytosis is increased in these glucose independent cells, and is regulated by phosphoinositide 3-kinase (PI3K) activation of Rac-Pak signaling. Furthermore, inhibition of Rac-dependent macropinocytosis blocks glucose-independent proliferation. We find that degradation of internalized protein produces amino acids, including alanine, which generates TCA cycle and glycolytic intermediates in the absence of glucose. In this process, the conversion of alanine to pyruvate by alanine transaminase 2 (ALT2) is critical for survival during glucose starvation. Collectively, Rac driven macropinocytosis of extracellular protein is an adaptive metabolic pathway used by a subset of lung cancers to survive states of glucose deprivation, and may serve as a potential drug target for cancer therapy.

The Central Role of PI3K in Cell Signaling Phosphatidylinositol 3-kinase (PI3K) is involved in multiple cell processes, including insulin signaling, cell growth, immunity, and brain development. It...

The recent end of the Human Terrain System run by the US Army has brought to a close one of the most recent insidious challenges to the field of anthropology. But while the anthropological elements of the programme have been well documented, and contested, its historical roots have not. The HTS and cultural knowledge apparatus of the American defence establishment draw deeply upon the wells of British scholar administrators of the nineteenth century. Of particular importance, especially in regard to Afghanistan, is Mountstuart Elphinstone who published the foundational text about the country, An account of the Kingdom of Kabul, 200 years ago. But unlike the HTS and similar programmes run by other Western governments which treat culture as a kind of technical aspect of governance, Elphinstone appreciated the importance - and legitimacy - of local politics.

SUMMARY Insulin stimulates the conversion of phosphatidylino-sitol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), which mediates downstream cellular responses. PI(4,5)P2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phos-phate 4-kinases (PIP4Ks). Here, we show that the loss of PIP4Ks (PIP4K2A, PIP4K2B, and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P2 and a concomitant increase in insulin-stimulated production of PI(3,4,5)P3. The reintroduction of either wild-type or kinase-dead mutants of the PIP4Ks restored cellular PI(4,5)P2 levels and insulin stimulation of the PI3K pathway, suggesting a catalytic-independent role of PIP4Ks in regulating PI(4,5)P2 levels. These effects are explained by an increase in PIP5K activity upon the deletion of PIP4Ks, which normally suppresses PIP5K activity through a direct binding interaction mediated by the N-terminal motif VMLϕFPDD of PIP4K. Our work uncovers an allosteric function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3 and suggests that the pharmacological depletion of PIP4K enzymes could represent a strategy for enhancing insulin signaling., Graphical Abstract, In Brief PI(4,5)P2 is produced by both phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Wang et al. report an allosteric function of a conserved N-terminal motif of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5) P3. This non-catalytic role has implications for the development of PIP4K targeted therapies.

Patient-derived culture models enable assessment of drug sensitivity and can connect personalized genomics with therapeutic options. However, their clinical translation is constrained by limited fidelity. We outline how the physical microenvironment regulates cell metabolism and describe how engineered culture systems could enhance the predictive power for precision medicine.

The cancer anorexia cachexia syndrome is a systemic metabolic disorder characterized by the catabolism of stored nutrients in skeletal muscle and adipose tissue that is particularly prevalent in nonsmall cell lung cancer (NSCLC). Loss of skeletal muscle results in functional impairments and increased mortality. The aim of the present study was to characterize the changes in systemic metabolism in a genetically engineered mouse model of NSCLC. We show that a portion of these animals develop loss of skeletal muscle, loss of adipose tissue, and increased inflammatory markers mirroring the human cachexia syndrome. Using noncachexic and fasted animals as controls, we report a unique cachexia metabolite phenotype that includes the loss of peroxisome proliferator-activated receptor-α (PPARα) -dependent ketone production by the liver. In this setting, glucocorticoid levels rise and correlate with skeletal muscle degradation and hepatic markers of gluconeogenesis. Restoring ketone production using the PPARα agonist, fenofibrate, prevents the loss of skeletal muscle mass and body weight. These results demonstrate how targeting hepatic metabolism can prevent muscle wasting in lung cancer, and provide evidence for a therapeutic strategy.

The tumor suppressor PTEN dephosphorylates PIP3 to inhibit PI3K signaling in cells. Altering PTEN intracellular signaling can therefore significantly affect cell behavior. Two novel mechanisms of PTEN regulation including the secretion and entry of the translational variant PTEN-L, and enzymatic inhibition by the interacting protein P-REX2, have been shown to modulate PI3K signaling, cellular proliferation and survival, and glucose metabolism. Here, we review the methods used to identify and validate the existence of both PTEN-L and the P-REX2–PTEN complex, to determine their effects on PTEN phosphatase activity, and to examine their role in cellular physiology.

From the invention of imperial authority along the North-West Frontier of British India, subjects were divided between the “civilized” inhabitants populating the cultivated plains and the “wild tribes” living in the hills. The problem of governing this latter group, the “independent tribes,” proved a vexed one for the British Raj. The mechanism developed by imperial administrators to manage the frontier inhabitants was the Frontier Crimes Regulation (FCR), first promulgated in 1872 and still in effect today. The FCR was designed to exclude the Frontier's inhabitants from the colonial judiciary, and more broadly the colonial sphere, encapsulating them in their own colonially sanctioned “tradition.” Exploring the use of the FCR as an instrument of governance from its first inception into the twentieth century, this article argues that it was key to shaping the nature of frontier rule, which in turn shaped the very nature of the colonial state itself.

Basal-like breast cancers (BBCs) are enriched for increased EGFR expression and decreased expression of PTEN. We found that treatment with metformin and erlotinib synergistically induced apoptosis in a subset of BBC cell lines. The drug combination led to enhanced reduction of EGFR, AKT, S6 and 4EBP1 phosphorylation, as well as prevented colony formation and inhibited mammosphere outgrowth. Our data with other compounds suggested that biguanides combined with EGFR inhibitors have the potential to outperform other targeted drug combinations and could be employed in other breast cancer subtypes, as well as other tumor types, with activated EGFR and PI3K signaling. Analysis of BBC cell line alterations led to the hypothesis that loss of PTEN sensitized cells to the drug combination which was confirmed using isogenic cell line models with and without PTEN expression. Combined metformin and erlotinib led to partial regression of PTEN-null and EGFR-amplified xenografted MDA-MB-468 BBC tumors with evidence of significant apoptosis, reduction of EGFR and AKT signaling, and lack of altered plasma insulin levels. Combined treatment also inhibited xenografted PTEN null HCC-70 BBC cells. Measurement of trough plasma drug levels in xenografted mice and a separately performed pharmacokinetics modeling study support possible clinical translation.

Makers of Modern Asia, edited by Ramachandra Guha. Cambridge, The Belknap Press of Harvard University Press, 2014. 385 pp. $29.95 US (cloth). In his volume Makers of Modern Asia, Ramachandra Guha has brought together an impressive assembly of scholars, journalists, and public intellectuals tasked with authoring a series of biographical essays about Asia's major leading political figures of the twentieth century. The list of figures, while in many ways predictable, is notable for its inclusivity. In it, the reader finds three Indian (Mohandas Gandhi, Jawaharlal Nehru, and Indira Gandhi) and four Chinese (Chiang Kai-shek, Mao Zedong, Zhou Enlai, and Deng Xiaoping) leaders, as well as one each from Vietnam (Ho Chi Minh), Indonesia (Sukarno), Singapore (Lee Kuan Yew), and Pakistan (Zulfikar Ali Bhutto). The list reads like a roster of the veritable heavyweights of Asian statesmen (and a token woman) of the recent past. In keeping with the volume's theme of "makers," nearly all the selected individuals were key movers in the nationalist movements that gained independence (de jure and de facto) throughout Asia during the middle decades of the century. Makers of Modern Asia begins with a short introductory essay authored by Guha himself, where he lays out the structure and logic of the book. The book's aim is to bring to the fore the "now somewhat obscured history of agitation and consolidation that created unified, stable (or more or less stable) nation-states out of fragmented territories and fractious social groups" (4). The work, then, is most decidedly that of political biography. As such, it returns to a version of history rather long passe within the profession, namely that of "big-man" history. Yet the authors use this form to reflect on wider social and political issues that their subjects both shaped and were beholden to. Taken individually, the eleven essays offer contributions of somewhat uneven character. Some, such as James R. Rush's chapter on Sukarno and Odd Arne Westad's on Deng Xiaoping, provide a chronological biography of their subject from birth to death. Others, such as Sophie Quinn-Judge's contribution on Ho-Chi Mirth, more tightly focus on particular episodes of the individual's life, in this case the years between the end of WWII and the outbreak of the Second Indo-China War. Consequently, the essays fail to set a collective tone for the volume, as each reflects the individual voice of its author. Some take a relatively light tack vis-a-vis the details of the historical narrative, while others provide what is at times an overwhelming wealth of information. …

Phosphoinositide 3-kinase (PI3K) activity is stimulated by diverse oncogenes and growth factor receptors, and elevated PI3K signaling is considered a hallmark of cancer. Many PI3K pathway-targeted therapies have been tested in oncology trials, resulting in regulatory approval of one isoform-selective inhibitor (idelalisib) for treatment of certain blood cancers, and a variety of other agents at different stages of development. In parallel to PI3K research by cancer biologists, investigations in other fields have uncovered exciting and often unpredicted roles for PI3K catalytic and regulatory subunits in normal cell function and in disease. Many of these functions impinge upon oncology by influencing the efficacy and toxicity of PI3K-targeted therapies. Here we provide a perspective on the roles of class I PI3Ks in the regulation of cellular metabolism and in immune system functions, two topics closely intertwined with cancer biology. We also discuss recent progress developing PI3K-targeted therapies for treatment of cancer and other diseases.