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Supplementary Figure S1. Generation of GEMM-derived cell lines. Supplementary Figure S2. QPCR and Western blot analyses of DTYMK expression in 634 cells upon indicated shDtymks transduction. Supplementary Figure S3. Lkb1-wt 634 and Lkb1-null t4 cells transduced with shGFP, shDtymk-1 or shDtymk-3 and then cultured with or without additional 100 microM dTTP in medium for 4 days. Supplementary Figure S4. Lkb1-wt (634, 855, and 857) and Lkb1-null (t2, t4, and t5) cell lines (A) and human LKB1-wt (H358 and Calu-1) and LKB1-deficient H2122 and A549) NSCLC cell lines (B) were treated with CHEK1 inhibitors, AZD7762 and CHIR124, for the indicated dose and time, and then lysed for Western blot analysis of gammaH2AX. Supplementary Figure S5. Immunohistochemical analyses assessing gammaH2AZ. Supplementary Figure S6. QPCR and Western blot analyses of DTYMK expression in LKB1-wt Calu-1 NSCLC cells. Supplementary Figure S7. A schematic model is proposed based on our current data. Supplementary Figure S8. QPCR analyses of Dtymk (A) and Chek1 (B) transcript levels in Lkb1-wt (634, 855, and 857) and Lkb1-null (t2, t4, and t5) cell lines. Supplementary Figure S9. QPCR analysis of TK1 expression in 634 cells upon the indicated shTK1 transduction for 3 days. Supplementary Figure S10. Gene expression levels of DTYMK in normal lung vs. lung adenocarcinoma from the Oncomine Cancer Microarray database.

The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is adjacent to the CDKN2A tumor suppressor and codeleted with CDKN2A in approximately 15% of all cancers. Previous attempts to target MAT2A with small-molecule inhibitors identified cellular adaptations that blunted their efficacy. Here, we report the discovery of highly potent, selective, orally bioavailable MAT2A inhibitors that overcome these challenges. Fragment screening followed by iterative structure-guided design enabled >10 000-fold improvement in potency of a family of allosteric MAT2A inhibitors that are substrate noncompetitive and inhibit release of the product, S-adenosyl methionine (SAM), from the enzyme's active site. We demonstrate that potent MAT2A inhibitors substantially reduce SAM levels in cancer cells and selectively block proliferation of MTAP-null cells both in tissue culture and xenograft tumors. These data supported progressing AG-270 into current clinical studies (ClinicalTrials.gov NCT03435250).

Discovery of targeted therapies that selectively exploit the genetic inactivation of specific tumor suppressors remains a major challenge. This includes the prevalent deletion of the CDKN2A/ MTAP locus, which was first reported nearly 40 years ago. The more recent advent of RNA interference and functional genomic screening technologies led to the identification of hidden collateral lethalities occurring with passenger deletions of MTAP in cancer cells. In particular, small-molecule inhibition of the type II arginine methyltransferase PRMT5 and the S-adenosylmethionine-producing enzyme MAT2A each presents a precision medicine approach for the treatment of patients whose tumors have homozygous loss of MTAP. In this review, we highlight key aspects of MTAP, PRMT5, and MAT2A biology to provide a conceptual framework for developing novel therapeutic strategies in tumors with MTAP deletion and to summarize ongoing efforts to drug PRMT5 and MAT2A.

Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers., Cancer cells are metabolically adaptable and the identification of specific vulnerabilities is challenging. Here the authors identify a subset of neuroendocrine cell lines exquisitely sensitive to inhibition of SQLE, an enzyme in the cholesterol biosynthetic pathway, due to the toxic accumulation of pathway intermediate squalene.

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.

This paper examines the perceptions of behavior-analytic professionals holding credentials through the Behavior Analyst Certification Board® (BACB®)-including Board Certified Behavior Analysts-Doctoral™, Board Certified Behavior Analysts®, and Board Certified Assistant Behavior Analysts®-regarding multiculturalism and diversity issues in their graduate training, fieldwork, and supervision. This paper predominantly focuses on future directions for improving graduate training, fieldwork, and supervision requirements in the field of applied behavior analysis (ABA) to produce more culturally competent professionals. Results from a preliminary survey of BACB® certificants (N = 575) are included to provide a context for recommendations on how to move the field of ABA forward to enhance the training and preparation of future credentialed professionals.

Isocitrate dehydrogenase (IDH) 1 and 2 mutations result in overproduction of D-2-hydroxyglutarate (2-HG) and impaired cellular differentiation. Ivosidenib, a targeted mutant IDH1 (mIDH1) enzyme inhibitor, can restore normal differentiation and results in clinical responses in a subset of patients with mIDH1 relapsed/refractory (R/R) acute myeloid leukemia (AML). We explored mechanisms of ivosidenib resistance in 174 patients with confirmed mIDH1 R/R AML from a phase 1 trial. Receptor tyrosine kinase (RTK) pathway mutations were associated with primary resistance to ivosidenib. Multiple mechanisms contributed to acquired resistance, particularly outgrowth of RTK pathway mutations and 2-HG–restoring mutations (second-site IDH1 mutations, IDH2 mutations). Observation of multiple concurrent mechanisms in individual patients underscores the complex biology of resistance and has important implications for rational combination therapy design. This trial was registered at www.clinicaltrials.gov as #NCT02074839

The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.

SummaryHomozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers.

MAT2A (methionine adenosyltransferase 2 alpha) is a critical enzyme within the methionine salvage pathway responsible for generating the universal methyl group donor, S-adenosyl methionine (SAM). We have developed a first-in-class small molecule inhibitor of MAT2A, AG-270, currently in a phase 1 clinical study (ClinicalTrials.gov NCT03435250) for the treatment of patients with solid tumors or lymphomas with MTAP (methylthioadenosine phosphorylase) deletion. The MTAP gene is deleted in approximately 15% of all human cancers, including non-small cell lung cancer (NSCLC; ~15-25%), pancreatic (~25%) and esophageal (~30%) cancer, and glioblastoma (~50%). To prioritize candidate combination partners for AG-270, a cell-based in vitro screening approach was employed using MTAP-null cell lines, in which AG-270 was combined with standard-of-care (SOC) agents as well as agents targeting pathways with hypothesized mechanistic links to MAT2A. Some of the best performing enhancers from this screen included paclitaxel (and docetaxel, using orthogonal screens) and gemcitabine. To assess the robustness of these combination findings in clinically relevant in vivo models, a series of patient-derived xenograft (PDX) experiments was undertaken to evaluate tolerability and efficacy in mice. Results demonstrated that AG-270, when combined with taxanes (paclitaxel/docetaxel) or gemcitabine, was well tolerated using SOC plasma exposures less than or equal to those achieved in patients. Importantly, combining AG-270 with taxanes and gemcitabine yielded additive-to-synergistic anti-tumor activity, with the docetaxel combination yielding 50% complete tumor regressions (CRs) in 2-3 PDX models. To study the mechanism of action, MAT2A was inhibited in vitro within HCT-116 MTAP −/− and wild-type cells, and we observed RNA splicing changes (via detained introns) altering genes involved in cell cycle regulation and DNA damage response, with a more pronounced effect found in the MTAP −/− genetic setting. Moreover, detained introns involving these same two pathways were modulated in MTAP −/− NSCLC PDX models treated with AG-270. Taken together, these data suggest AG-270 complements the known mechanism of action of taxanes and gemcitabine, and leads to enhanced DNA damage and inhibition of cellular proliferation. This work has helped identify a therapeutic strategy of combining AG-270 with taxanes and gemcitabine, which is currently being explored in an ongoing phase 1 clinical trial (NCT03435250). Page 1 of 1 Citation Format: Marc L. Hyer, Peter Kalev, Mark Fletcher, Chi-Chao Chen, Elia Aguado-Fraile, Everton Mandley, Sheila Newhouse, Max Lein, Raj Nagaraja, Yesim Tuncay, Josh Murtie, Kevin M. Marks, Katya Marjon. The MAT2A inhibitor, AG-270, combines with both taxanes and gemcitabine to yield enhanced anti-tumor activity in patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3090.

Methylthioadenosine phosphorylase (MTAP) is the enzyme in the methionine salvage pathway that metabolizes the byproduct of polyamine biosynthesis, 5′-methylthioadenosine (MTA). The MTAP gene is deleted in approximately 15% of all human cancers and results in accumulation of MTA that partially inhibits protein arginine methyl transferase 5 (PRMT5) function. Recent studies have demonstrated that loss of the MTAP gene sensitizes cancer cells to genetic depletion of PRMT5 and the upstream metabolic enzyme methionine adenosyltransferase 2 alpha (MAT2A). We have developed a first-in-class, highly potent, orally bioavailable MAT2A inhibitor, AG-270, which is currently under investigation in a phase 1 clinical trial (ClinicalTrials.gov NCT03435250). AG-270 selectively inhibits the growth of HCT116 MTAP-/- cells compared to HCT116 MTAP+/+ cells in vitro and in vivo. We utilized RNA sequencing and proteomic approaches to study the underlying mechanism of action for AG-270. The results of these analyses demonstrated that AG-270 treatment induces substantial splicing changes in an MTAP selective manner. The majority of these abnormal splicing events were detained introns, which included genes involved in cell cycle regulation and DNA damage response. Moreover, AG-270 treatment resulted in accumulation of DNA damage and increased mitotic defects in HCT116 MTAP-/- cells. Furthermore, we demonstrated substantially advantageous combination benefits between AG-270 and anti-mitotic taxanes in in vitro cell lines and in in vivo patient-derived xenograft models of non-small cell lung, pancreatic and esophageal cancers. These findings uncover relevant combination strategies targeting MTAP-deleted malignancies, and provide preclinical mechanistic proof-of-concept to explore such combinations in the clinical setting. Citation Format: Peter Kalev, Marc L Hyer, Mark Fletcher, Peili Zhang, Elia Aguado-Fraile, Everton Mandley, Yesim Tuncay, Zenon Konteatis, Jeremy Travins, Kevin M Marks, Katya Marjon. Mitotic defects induced by MAT2A inhibitors guides translational drug combination strategies with AG-270 and taxanes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B115. doi:10.1158/1535-7163.TARG-19-B115

Background: Homozygous deletion of MTAP, the gene encoding the metabolic enzyme methylthioadenosine phosphorylase, occurs in ~15% of human malignancies. Tumor cells with this deletion are selectively vulnerable to decreases in the methyl donor S-adenosylmethionine (SAM). AG-270 is a first-in-class, oral, potent, reversible inhibitor of methionine adenosyltransferase 2A (MAT2A), the key enzyme responsible for SAM synthesis. We report preliminary results from an ongoing, first-in-human, phase 1 trial of AG-270 (ClinicalTrials.gov Identifier: NCT03435250). Aims: The primary objective of this study is to determine the maximum tolerated dose (MTD) of AG-270. Secondary objectives include safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. Methods: Eligibility requires homozygous deletion of cyclin dependent kinase inhibitor 2A (CDKN2A) in the patient’s tumor (as MTAP is usually co-deleted with CDKN2A), or loss of MTAP by IHC. Patients receive AG-270 daily in 28-day cycles, with intensive PK/PD sampling after the first dose and after 2 weeks of treatment. Paired tumor biopsies are collected at baseline and at the end of cycle 1. Disease evaluation is performed every 2 cycles. Results: As of 20 May 2019, 39 patients had been treated with AG-270: 50 mg once daily (QD; n=3), 100 mg QD (n=7), 150 mg QD (n=6), 200 mg QD (n=11), 400 mg QD (n=6), or 200 mg twice daily (BID; n=6). AG-270 was well absorbed. Plasma concentrations increased in a dose-proportional manner except at 400 mg QD, where exposure was lower than anticipated. The geometric mean area under the curve from 0-24 h at steady state (AUC0-24,ss) in the QD cohorts ranged from 33200 to 199085 ng*h/mL, and the geometric mean AUC0-24,ss in the 200 mg BID cohort was 254616 ng*h/mL. The median half-life of AG-270 ranged from 16.1 to 38.4 h. Decreases in plasma [SAM] were exposure-dependent. After 2 weeks of dosing, maximal reductions in plasma [SAM] ranged from 51% to 71% across the tested cohorts. Analysis of 9 paired tumor biopsies by IHC showed decreases in levels of symmetrically di-methylated arginine (SDMA) residues, consistent with MAT2A inhibition; the average H-score reduction compared to baseline was 36.4% [-98.8%, +21.4%]. Asymptomatic, exposure-dependent increases in unconjugated bilirubin were observed starting at 100 mg QD, consistent with the known potential of AG-270 to inhibit UGT1A1. Three patients (at 100 mg QD, 150 mg QD, and 200 mg BID) developed grade 2 and 3 diffuse erythematous rashes during the second week of dosing that resolved within 1 week of stopping treatment. Exposure-dependent, reversible decreases in platelet counts were first observed at 200 mg QD and were grade 3 and 4 in severity at 200 mg BID. Two patients treated at 200 mg BID developed reversible but dose-limiting grade 3 and 4 increases in liver enzymes. The MTD of AG-270 is 200 mg QD. An unconfirmed partial response has been observed in a patient with a high-grade neuroendocrine carcinoma of the lung. Seven patients have achieved radiographically confirmed stable disease of 2.0 to 9.9 months’ duration. Conclusions: AG-270 causes reductions in plasma [SAM] and in tumor SDMA levels at well-tolerated doses. This trial will next evaluate the combination of AG-270 with taxane-based chemotherapy, given preclinical data demonstrating enhanced antitumor activity with AG-270 and taxanes in MTAP-deleted cancer models. Citation Format: Rebecca S Heist, Mrinal M Gounder, Sophie Postel-Vinay, Frederick Wilson, Elena Garralda, Khanh Do, Geoffrey I Shapiro, Patricia Martin-Romano, Gerburg Wulf, Michael Cooper, Caroline Almon, Salah Nabhan, Varsha Iyer, Yanwei Zhang, Kevin Marks, Elia Aguado-Fraile, Frank Basile, Keith Flaherty, Howard A Burris. A phase 1 trial of AG-270 in patients with advanced solid tumors or lymphoma with homozygous MTAP deletion [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR03. doi:10.1158/1535-7163.TARG-19-PR03

Nature 547, 109–113 (2017); doi:10.1038/nature22964 In this Letter, there are errors in Extended Data Figs 5, 8 and 9, and the affiliation of an author. The affiliations for author Violeta Serra should include number 2 (CIBERONC, Instituto de Salud Carlos III, C/ Monforte de Lemos 3-5, Pabellon 11, Planta 0, 28029 Madrid, Spain).

While deletions of the p16/CDKN2A tumor suppressor were first discovered more than 30 years ago, therapeutics that selectively target such tumors have proven elusive. Recent work utilizing functional genomics has identified a synthetic lethal vulnerability that arises due to co-deletion of the adjacent metabolic gene, methylthioadenosine phosphorylase (MTAP). Loss of MTAP in these tumors leads to an accumulation of MTAP substrate 5'-methylthioadenosine (MTA), which partially inhibits the arginine methyltransferase PRMT5 and sensitizes tumors to shRNA-mediated depletion of PRMT5 and the upstream metabolic enzyme, methionine adenosyltransferase 2 alpha (MAT2A). To investigate the therapeutic potential of this finding, we utilized a biophysical binding screen followed by iterative structure-guided design to make the first highly potent, selective, and orally bioavailable inhibitors of MAT2A. MAT2A inhibitor treatment leads to potent inhibition of the growth of HCT116 MTAP-/- cells while sparing isogenic HCT116 MTAP+/+ cells. Tumor xenograft studies similarly demonstrated MTAP-selective growth inhibition in HCT116 MTAP-/- tumors compared to isogenic HCT116 MTAP+/+ tumors. Further, MTAP-deletion correlated with MAT2A inhibitor efficacy across a panel of >300 cell lines in vitro, and MAT2A inhibitor treatment was efficacious in a variety of MTAP-deleted patient-derived xenografts in vivo. Having demonstrated that potent MAT2A inhibitors selectively block the proliferation of MTAP-deleted cells and tumors, we sought to investigate the mechanism by which these effects arise. Using methylation proteomics we noted that MAT2A inhibitor treatment leads to selective inhibition of PRMT5 methylation activity in MTAP-deleted cancers in vitro and in vivo. RNA-seq analyses revealed that MAT2A inhibition leads to substantial defects in RNA splicing in MTAP-deleted cancers, consistent with published findings that PRMT5-mediated methylation of splicing complex proteins is critical for their function. MAT2A inhibitor treatment led to a substantial increase in detained introns, which were enriched in genes involved in cell cycle regulation and DNA damage response, thus implicating dysregulated splicing in the antiproliferative effects of MAT2A inhibition in MTAP-deleted cancer cells. Furthermore, we demonstrated substantial drug-drug synergy between MAT2A inhibitors and select agents inhibiting cell cycle progression or DNA repair. Importantly we validated key combination findings in vivo, including demonstration of synergy with the MAT2A inhibitor AG-270 and anti-mitotic taxanes. AG-270 is the first MAT2A inhibitor to enter clinical development and is under investigation in a Phase I trial that is currently enrolling patients with MTAP-deleted solid tumors (NCT03435250). Our findings suggest clinically-applicable combination strategies which may further enhance the efficacy of AG-270 in malignancies with this genetic lesion. Citation Format: Katya Marjon, Peter Kalev, Marc Hyer, Mark Fletcher, Peili Zhang, Elia Aguado-Fraile, Everton Mandley, Zenon Konteatis, Jeremy Travins, Kevin Marks. Targeting MAT2A in CDKN2A/MTAP-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2714.

SummaryProliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.

Mutations in tumor suppressor genes are a hallmark of cancer and contribute substantially to the molecular pathogenesis of the disease. Particularly prevalent are homozygous deletions of the CDKN2A tumor suppressor locus on chromosome 9p21, which occur in at least 15% of all cancer and lead to loss of the dual tumor suppressors p16(INK4A) and p14(ARF). Targeted therapies selective for loss of CDKN2A have proven elusive, and it may be necessary to identify alternate approaches to target tumors with this deletion. Notably, Chr9p21 deletions frequently involve co-deletion of genes proximal to CDKN2A. Foremost among these is the methylthioadenosine phosphorylase (MTAP) gene, which resides within 100 kb of CDKN2A, and is co-deleted in 80-90% of tumors with CDKN2A deletion. We sought therefore to evaluate whether the frequent co-deletion of the adjacent metabolic gene methylthioadenosine phosphorylase (MTAP) creates targetable vulnerabilities. We conducted an shRNA screen in isogenic cells that varied only in their MTAP-deletion status, and identified the metabolic enzyme, methionine adenosyltransferase II, alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss, and biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deletion leads to reduced basal PRMT5 methylation activity and increased sensitivity to PRMT5 targeting. MAT2A produces PRMT5 substrate S-adenosylmethionine (SAM), and targeting of MAT2A reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Further, this vulnerability extends to PRMT5 co-complex proteins such as the atypical kinase Rio Kinase 1 (RIOK1). Thus, MAT2A, PRMT5, and RIOK1 comprise an unappreciated axis of targets that is selectively vulnerable in CDKN2A/MTAP-deleted tumors. Unique biochemical features of PRMT5 create this vulnerability, which presents a promising new avenue for targeted therapy. Citation Format: Kevin Marks. Targeting cancers with deletion of the p16/p14 tumor suppressor locus. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA25.

Cancer metabolism is an emerging field that offers promise in revealing new targets and strategies to treat tumors. Here, we describe several of the major advances in the understanding of how cancer cells rewire their metabolic circuitry and how these insights might be used to develop new drugs. We contrast how gain-of-function mutations in tyrosine kinases and metabolic enzymes such as isocitrate dehydrogenase promote tumorigenesis. While such recurrent mutations have not been described for the vast majority of metabolic enzymes, these enzymes are still potential targets in their own right, or are used by cancer cells in ways that can expose other vulnerabilities. We illustrate how the ability to study metabolism in molecular detail through sophisticated nutrient labeling studies has allowed connections to be made between cancer metabolism and cell signaling, epigenetics, as well as pathways targeted by standard-of-care chemotherapeutics.

In HL-60 human myeloblastic leukemia cells, retinoic acid is known to cause cFMS, RAF, MEK, and ERK2 dependent myeloid cell differentiation and G0 arrest associated with RB tumor suppressor protein hypophosphorylation, implicating receptor tyrosine kinase signal transduction in propelling these retinoic acid-induced cellular effects. Furthermore, ectopic expression of polyoma middle T antigen, which activates similar early signal transduction molecules as PDGF class receptors such as cFMS, accelerates these retinoic acid-induced effects. To determine if this depends on middle T's ability to activate PLCgamma, PI-3 kinase, and src-like kinases, stable transfectants of HL-60 cells expressing either the polyoma middle T dl23 mutant, which is defective for PLCgamma and PI-3 kinase activation, or the Delta205 mutant, which in addition has greatly attenuated src-like kinase activation ability, were created and compared to wild-type middle T-transfected HL-60. The transgenes were under control of the retinoic acid (or 1, 25-dihydroxy vitamin D3) inducible Moloney murine leukemia virus LTRs. Expression of the dl23 or Delta205 mutant accelerated retinoic acid-induced cell differentiation. The effects of the mutants were comparable to those of the wild-type middle T. Likewise, retinoic acid-induced G0 arrest of mutant transfected cells and wild-type middle T transfected cells was similar. The same was true for 1, 25-dihydroxy vitamin D3-induced monocytic differentiation as for retinoic acid-induced myeloid differentiation. The mutants did not cause the same slight shortening of the cell cycle as wild-type middle T. Both the mutants and the wild-type middle T caused a similar increase in the cellular basal level of activated ERK2 MAPK. Since retinoic acid increases ERK2 activation, which is necessary for differentiation, the data suggest that mutant and wild-type middle T enhanced the retinoic acid effects by increasing basal levels of ERK2 activation. Consistent with this, the polyoma-induced foreshortening of the time for differentiation coincided with the time for retinoic acid to significantly increase ERK2 activation. As in wild-type HL-60, retinoic acid induced the early down-regulation of RXRalpha in mutant transfectants similar to wild-type middle T transfectants, consistent with no loss or gain of relevant functions due to the mutations. In contrast, vitamin D3 did not down-regulate RXRalpha in HL-60 or transfectants. Polyoma middle T and these transformation-defective mutants thus enhanced ERK2 activation to have an early effect in promoting retinoic acid-induced differentiation without a strong dependence on activating PLCgamma, PI-3 kinase, or src-like kinase.

The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase, which coordinates cell growth, polarity, motility, and metabolism. In non–small cell lung carcinoma, LKB1 is somatically inactivated in 25% to 30% of cases, often concurrently with activating KRAS mutations. Here, we used an integrative approach to define novel therapeutic targets in KRAS-driven LKB1-mutant lung cancers. High-throughput RNA interference screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase (DTYMK), which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling showed that Lkb1-null cells had a striking decrease in multiple nucleotide metabolites as compared with the Lkb1–wild-type cells. Thus, LKB1-mutant lung cancers have deficits in nucleotide metabolism that confer hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors. Significance: Using cell lines derived from the lung cancers occurring in genetically engineered mice, we conducted an integrative genome-wide short hairpin RNA and metabolite screen to identify DTYMK as a potential therapeutic target in Kras/Lkb1–mutant lung cancer. We believe that DTYMK is tractable for the development of novel therapeutics, and show an integrative approach to target identification that reduces false-positive candidates and should have broad applicability for the development of targeted therapeutics. Cancer Discov; 3(8); 870–9. ©2013 AACR. See related commentary by Marcus and Khuri, p. 843 This article is highlighted in the In This Issue feature, p. 826

Homozygous deletions of the CDKN2A tumor suppressor locus occur in approximately 15% of all cancer and represent a substantial therapeutic need(1). Targeted therapies selective for loss of CDKN2A have proven elusive, so we sought to evaluate whether the frequent co-deletion of the adjacent metabolic gene methylthioadenosine phosphorylase (MTAP) leads to collateral vulnerabilities. We conducted an shRNA screen in isogenic cells that vary only in their MTAP-deletion status, and identified an axis of targets that become vulnerable upon MTAP loss. Central in this axis is the arginine methyltransferase, PRMT5. Targeting PRMT5 with doxycycline-inducible shRNA impaired the growth of MTAP-deleted HCT116 colon carcinoma cells without impact on the growth of MTAP-wt HCT116 cells. Having identified this synthetic lethality between PRMT5 and MTAP, we sought next to elucidate the mechanistic basis for this relationship. Untargeted metabolomics revealed that MTA, the substrate of the MTAP enzyme reaction, accumulates dramatically in MTAP-deleted cancers. Biochemical profiling across the methyltransferase enzyme family revealed that MTA is a potent and selective inhibitor of PRMT5. Consistent with this in vitro finding, MTA accumulation in MTAP-deleted cancers led to reduced basal PRMT5 methylation. This selective vulnerability extends beyond PRMT5 to methionine adenosyltransferase 2, a (MAT2A), the metabolic enzyme upstream of PRMT5. MAT2A was the top hit in the shRNA screen, and targeting of MAT2A with inducible shRNA led to selective inhibition of growth in MTAP-deleted HCT116 cells in vitro and in vivo. Targeting of MAT2A led to substantial reduction of PRMT5 methylation activity in MTAP-deleted cells, indicating that PRMT5 activity is controlled by levels of substrate SAM and inhibitory metabolite MTA. To assess the therapeutic relevance of this axis in settings with endogenous deletion of the CDKN2A/MTAP locus, we depleted MAT2A in a panel of cancer cell lines and observed selective growth inhibition in the lines with endogenous deletion of CDKN2A/MTAP. Lastly, we noted that RIO Kinase 1 (RIOK1), a PRMT5-interacting protein(2), also scored in the shRNA screen. Doxycycline-inducible shRNA studies validated RIOK1 as an MTAP-selective target, and additional siRNA studies indicated that multiple PRMT5 co-complex members are selectively essential in MTAP-deleted cells. Thus, PRMT5 is the central node in an axis of targets that could be exploited therapeutically in cancers with deletion of CDKN2A/MTAP. References: 1 Beroukhim, R. et al. The landscape of somatic copy-number alteration across human cancers. Nature 463, 899-905, doi:10.1038/nature08822 (2010). 2 Guderian, G. et al. RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity. J. Biol. Chem. 286, 1976-1986, doi:10.1074/jbc.M110.148486 (2011). Citation Format: Kevin Marks. MTAP deletions in cancer create vulnerability to a MAT2A/PRMT5/RIOK1 axis. [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 LB-307.

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation[superscript 1, 2]. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes[superscript 3]. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets., Susan G. Komen Breast Cancer Foundation (Fellowship), Life Sciences Research Foundation (Fellowship), W. M. Keck Foundation, David H. Koch Cancer Research Fund, Alexander and Margaret Stewart Trust, National Institutes of Health (U.S.) (Grant CA103866)

Lymphangioleiomyomatosis (LAM) is a female predominant lung disease characterized by widely-distributed nodules of ‘LAM’ cells and progressive cyst formation that leads to respiratory failure. LAM cells typically have mutation or inactivation of TSC2, leading to mTORC1 activation. We previously demonstrated that estrogen promotes the survival of tuberin-deficient ELT3 cells both in vitro and in vivo, and that estrogen treatment of mice bearing ELT3 cells xenograft tumors promotes lung metastasis. This estrogen-induced cell survival and metastasis was inhibited in vivo by the MEK inhibitor CI-1040. To elucidate pro-survival events mediated by estrogen in tuberin-null cells, we compared Tsc2-null ELT3 cell xenograft tumors from estrogen or placebo-treated mice using expression profiling and proteomic analysis . Expression profiling of ELT3 cell xenograft tumors revealed that estrogen upregulates the expression of genes in lipid and amino acid metabolism pathways. Among these genes, the estrogen-induced expression of amino adipatetransferase (AADAT), which is involved in lysine degradation, has been confirmed by immunoblotting of tumor lysates. Using iTRAQ proteomics from ELT3 xenograft tumors, we identified 40 proteins that are significantly regulated by estrogen, including proteins involved in carbohydrate metabolism (creatine kinase), amino acid degradation (aspartate aminotransferase) and lipid metabolism (isocitrate dehydrogenase). Metabolic profiling by LC/MS/MS of TSC2-null angiomyolipoma-derived cells from a LAM patient (621-101 cells) showed a temporal effect of rapamycin (10 nM) on the accumulation of pentose phosphate pathway intermediates and glycolytic metabolites. Cellular amino acid levels were reduced after rapamycin treatment. Consistent with a metabolic switch occurring in LAM, we found, using IHC staining, that the tumor-associated PKM2 is abundantly expressed in ELT3 cell xenograft tumors, lung metastatic lesions from estrogen-treated mice bearing ELT3 cell xenograft tumors, and in human LAM cell nodules. Finally, using FDG-PET, we found that xenograft ELT3 cell tumors in estrogen-treated mice exhibited higher levels of uptake compared with placebo-treated mice. In conclusion, gene expression profiling indicated that estrogen enhances the expression of genes which products regulate glucose and amino acid metabolism. Proteomic analysis showed estrogen-regulated candidates involved in glycolysis, amino acid, and lipid metabolisms. Metabolomic screening revealed that cells lacking tuberin have a metabolic response to rapamycin treatment. FDG-PET imaging showed enhanced uptake in estrogen-treated xenograft tumors. Collectively, these data indicate that cellular metabolic alterations may contribute to the pathogenesis of LAM. Targeting metabolic regulators might have therapeutic benefit for LAM. Citation Format: Yang Sun, Chenggang Li, Kevin Marks, Erik Zhang, Tasha Morrison, Mi-Ae Park, Shuyan Wang, Simon Dillon, Manoj Bhasin, Towia Libermann, Edward Driggers, Victor Gerbaudo, Elizabeth Petri Henske, Jane Yu. Evidence from expression profiling, proteomics, metabolomics and FDG-PET of an estrogen-dependent metabolic switch in tuberin-null cells. [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 LB-129. doi:10.1158/1538-7445.AM2013-LB-129

It is well recognized that proliferating tumor cells invariably utilize aerobic glycolysis to support their high metabolic demands. Recent studies have further elucidated the paradoxical observation that this increase in glycolysis in proliferating tumor cells is often coupled with a molecular switch from the constitutively high activity PKM1 isoform to the modulated lower activity PKM2 isoform1,2, effectively constraining lower glycolysis at the last pyruvate kinase step. Here, we report an unanticipated link between glucose metabolism and amino-acid metabolic pathways in cancer cells. We characterized the biochemical and cellular effects of a small-molecule allosteric activator of the M2 isoform of pyruvate kinase (AGX-257) in various cancer cell types. We demonstrate that activation of PKM2 results in a metabolic rewiring of specific cancer cells that results in a profound dependency on the presence of the normally non-essential amino acid serine for continued cell proliferation. The induced serine auxotrophy by PKM2 activation is concomitant with reduced flux through the endogenous serine biosynthetic pathway and increased levels of high affinity serine transporters. PKM2 activators, possibly in combination with agents that modulate the cellular and extracellular serine pools, could form the basis for a new approach toward developing therapy for treating cancer. References: 1 Christofk, H. R. et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230-233 (2008). 2 Clower, C. V. et al. The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism. Proc Natl Acad Sci U S A 107, 1894-1899 (2010). 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 1002. doi:1538-7445.AM2012-1002

Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary human brain cancers. These mutations occur at a single amino acid residue of the IDH1 active site, resulting in loss of the enzyme's ability to catalyse conversion of isocitrate to alpha-ketoglutarate. However, only a single copy of the gene is mutated in tumours, raising the possibility that the mutations do not result in a simple loss of function. Here we show that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of alpha-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). Structural studies demonstrate that when arginine 132 is mutated to histidine, residues in the active site are shifted to produce structural changes consistent with reduced oxidative decarboxylation of isocitrate and acquisition of the ability to convert alpha-ketoglutarate to 2HG. Excess accumulation of 2HG has been shown to lead to an elevated risk of malignant brain tumours in patients with inborn errors of 2HG metabolism. Similarly, in human malignant gliomas harbouring IDH1 mutations, we find markedly elevated levels of 2HG. These data demonstrate that the IDH1 mutations result in production of the onco-metabolite 2HG, and indicate that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.

This paper discusses the definition and application of educational technology, how its development from a concept to a rational system in everyday use has affected the medical illustrator and what type of relationship educational technology and the medical illustrator will enjoy in the future.(The term ‘medical illustrator’ is used here to mean people working in medical art and graphic design. However, the views put forward have implications for all who come under the broader generic use of the term ‘medical illustrator’.)