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Supplementary Materials and Methods Supplementary References Tables S1 to S3 and legends Figures S1 to S4 and legends

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non–small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC.Significance:These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Supplemental Digital Content is Available in the Text. Dual leucine zipper kinase inhibition is a promising therapeutic strategy against chemotherapy-induced neurotoxicities including chemotherapy-induced peripheral neuropathy and chemotherapy-induced cognitive impairments., Chemotherapy-induced peripheral neuropathy (CIPN) and chemotherapy-induced cognitive impairments (CICI) are common, often severe neurotoxic side effects of cancer treatment that greatly reduce quality of life of cancer patients and survivors. Currently, there are no Food and Drug Administration-approved agents for the prevention or curative treatment of CIPN or CICI. The dual leucine zipper kinase (DLK) is a key mediator of axonal degeneration that is localized to axons and coordinates the neuronal response to injury. We developed a novel brain-penetrant DLK inhibitor, IACS′8287, which demonstrates potent and highly selective inhibition of DLK in vitro and in vivo. Coadministration of IACS′8287 with the platinum derivative cisplatin prevents mechanical allodynia, loss of intraepidermal nerve fibers in the hind paws, cognitive deficits, and impairments in brain connectivity in mice, all without interfering with the antitumor activity of cisplatin. The protective effects of IACS′8287 are associated with preservation of mitochondrial function in dorsal root ganglion neurons and in brain synaptosomes. In addition, RNA sequencing analysis of dorsal root ganglia reveals modulation of genes involved in neuronal activity and markers for immune cell infiltration by DLK inhibition. These data indicate that CIPN and CICI require DLK signaling in mice, and DLK inhibitors could become an attractive treatment in the clinic when coadministered with cisplatin, and potentially other chemotherapeutic agents, to prevent neurotoxicities as a result of cancer treatment.

Summary: The plasticity of a preexisting regulatory circuit compromises the effectiveness of targeted therapies, and leveraging genetic vulnerabilities in cancer cells may overcome such adaptations. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is characterized by oxidative phosphorylation (OXPHOS) deficiency caused by fumarate hydratase (FH) nullizyogosity. To identify metabolic genes that are synthetically lethal with OXPHOS deficiency, we conducted a genetic loss-of-function screen and found that phosphogluconate dehydrogenase (PGD) inhibition robustly blocks the proliferation of FH mutant cancer cells both in vitro and in vivo. Mechanistically, PGD inhibition blocks glycolysis, suppresses reductive carboxylation of glutamine, and increases the NADP+/NADPH ratio to disrupt redox homeostasis. Furthermore, in the OXPHOS-proficient context, blocking OXPHOS using the small-molecule inhibitor IACS-010759 enhances sensitivity to PGD inhibition in vitro and in vivo. Together, our study reveals a dependency on PGD in OXPHOS-deficient tumors that might inform therapeutic intervention in specific patient populations. : Loss-of-function genetics screen reveals a synthetically lethal interaction between OXPHOS inhibition and phosphogluconate dehydrogenase (PGD) inactivation. Sun et al. provide an example of targeting tumor metabolism in a genetically predefined context to maximize therapeutic impact and propose PGD as a therapeutic target for fumarate hydratase-deficient HLRCC. Keywords: synthetic lethality, PGD, OXPHOS, tumor metabolism, metabolic vulnerability, fumarate hydratase, redox homeostasis, functional genomics, hereditary leiomyomatosis renal cell carcinoma, pentose phosphate pathway

The disability, mortality and costs caused by non-vertebral osteoporotic fractures are enormous. Existing osteoporosis therapies are highly effective at reducing vertebral but not non-vertebral fractures. Cortical bone is a major determinant of non-vertebral bone strength. To identify novel osteoporosis drug targets, we phenotyped cortical bone of 3 366 viable mouse strains with global knockouts of druggable genes. Cortical bone thickness was substantially elevated in Notum−/− mice. NOTUM is a secreted WNT lipase and we observed high NOTUM expression in cortical bone and osteoblasts but not osteoclasts. Three orally active small molecules and a neutralizing antibody inhibiting NOTUM lipase activity were developed. They increased cortical bone thickness and strength at multiple skeletal sites in both gonadal intact and ovariectomized rodents by stimulating endocortical bone formation. Thus, inhibition of NOTUM activity is a potential novel anabolic therapy for strengthening cortical bone and preventing non-vertebral fractures., Bone Development: NOTUM inhibition stimulates bone formation NOTUM is an enzyme that inactivates WNT proteins (which play a key role in early tissue development), and inhibiting NOTUM has been found to increase the formation of endocortical bone (within the cortex, the hard exterior of bone) and enhance bone strength. Existing therapies for osteoporosis (condition causing bone to become weak and brittle) are effective in reducing vertebral, but not non-vertebral, fractures. A team headed by Robert Brommage at Lexicon Pharmaceuticals, Texas aimed to identify novel osteoporosis drug targets in mice. Following inhibition of NOTUM activity, the authors observed increased cortical bone thickness and strength at multiple skeletal sites through stimulation of endocortical bone formation. The team concluded that inhibiting NOTUM activity has good potential as a new therapeutic strategy and could be beneficial in preventing non-vertebral osteoporotic fractures.

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non–small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. Significance: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Src homology 2 domain-containing phosphatase (SHP2), a ubiquitously expressed non-receptor tyrosine phosphatase, plays a critical role in the regulation of the MAPK signaling pathway and cellular proliferation. Activating mutations in SHP2 are associated with the development of multiple malignancies including leukemia, lung cancer and neuroblastoma. In addition, SHP2 promotes the conversion of oncogenic KRAS to its active GTP-bound state and it’s inhibition can enhance efficacy of GDP-KRASG12C inhibitors as well as other MAPK pathway inhibitors (RAF, MEK and ERK) which have suboptimal clinical efficacy as single agents. As a result, inhibition of SHP2 through genetic manipulation or pharmacological means has been shown to suppress tumor growth and presents an attractive potential avenue for the treatment of malignancies as monotherapy or in combination with other MAPK/PI3K inhibitors. Here we describe BBP-398, a potent, orally bioavailable allosteric small molecule inhibitor of SHP2. BBP-398 displays high selectivity against other phosphatases, kinases, GPCRs, transporters and hERG. Predicted human PK properties show good oral bioavailability with half-life of ~12-16 hours enabling continuous daily dosing and optimal therapeutic index in combination with other targeted therapeutics. In cellular assays, BBP-398 demonstrates potent pERK/DUSP6 inhibition and loss of viability across a panel of cell lines with active MAPK signaling, such as mutant EGFR and KRASG12C. In vivo, BBP-398 strongly suppresses RAS-ERK signaling in RTK- or RAS-driven xenografts. In the EGFR-dependent non-small cell lung cancer (NSCLC) HCC827 and esophageal squamous cell carcinoma KYSE-520 xenograft models, BBP-398 drives dose dependent efficacy consistent with the level of target inhibition. Detailed analysis of tumor response shows that efficacy is driven by maintaining better than 50% inhibition of pERK for most of the dosing interval. In addition to its strong single agent activity, BBP-398 also leads to enhanced efficacy in vitro and in vivo when used in combination with targeted therapeutics against driver MAPK genetic alterations, such as KRAS, EGFR or MET. Combination targeting, such as with the GDP-KRASG12C inhibitor sotorasib in the NSCLC NCI-H358 xenograft model, or with the mutant EGFR inhibitor osimertinib in the HCC827 erlotinib resistant (ER) xenograft model, drives strong suppression of MAPK activity and results in tumor regressions. Collectively, these findings highlight that SHP2 inhibition is a promising molecular therapeutic strategy in cancer which can potentially strongly suppress tumor growth as a single agent or in combination with other MAPK pathway inhibitors. Given its preclinical properties and projected favorable clinical pharmacokinetic profile, BBP-398 is currently being evaluated in a Phase 1/1b trial in patients with advanced solid tumors (NCT04528836). Citation Format: James P. Stice, Sofia Donovan, Yuting Sun, Nancy Kohl, Barbara Czako, Faika Mseeh, Paul Leonard, Anna Wade, Justin Lim, Phil Jones, Eli Wallace, Kerstin Sinkevicius, Pedro Beltran. BBP-398, a potent, small molecule inhibitor of SHP2, enhances the response of established NSCLC xenografts to KRASG12C and mutEGFR inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P207.

LX2761 is a potent sodium/glucose cotransporter 1 inhibitor restricted to the intestinal lumen after oral administration. Studies presented here evaluated the effect of orally administered LX2761 on glycemic control in preclinical models. In healthy mice and rats treated with LX2761, blood glucose excursions were lower and plasma total glucagon-like peptide-1 (GLP-1) levels higher after an oral glucose challenge; these decreased glucose excursions persisted even when the glucose challenge occurred 15 hours after LX2761 dosing in ad lib-fed mice. Further, treating mice with LX2761 and the dipeptidyl-peptidase 4 inhibitor sitagliptin synergistically increased active GLP-1 levels, suggesting increased LX2761-mediated release of GLP-1 into the portal circulation. LX2761 also lowered postprandial glucose, fasting glucose, and hemoglobin A1C, and increased plasma total GLP-1, during long-term treatment of mice with either early- or late-onset streptozotocin-diabetes; in the late-onset cohort, LX2761 treatment improved survival. Mice and rats treated with LX2761 occasionally had diarrhea; this dose-dependent side effect decreased in severity and frequency over time, and LX2761 doses were identified that decreased postprandial glucose excursions without causing diarrhea. Further, the frequency of LX2761-associated diarrhea was greatly decreased in mice either by gradual dose escalation or by pretreatment with resistant starch 4, which is slowly digested to glucose in the colon, a process that primes the colon for glucose metabolism by selecting for glucose-fermenting bacterial species. These data suggest that clinical trials are warranted to determine if LX2761 doses and dosing strategies exist that provide improved glycemic control combined with adequate gastrointestinal tolerability in people living with diabetes.

Thirty-six patients with type 2 diabetes mellitus (T2DM) were randomized 1:1:1 to receive a once-daily oral dose of placebo or 150 or 300 mg of the dual SGLT1/SGLT2 inhibitor LX4211 for 28 days. Relative to placebo, LX4211 enhanced urinary glucose excretion by inhibiting SGLT2-mediated renal glucose reabsorption; markedly and significantly improved multiple measures of glycemic control, including fasting plasma glucose, oral glucose tolerance, and HbA(1c); and significantly lowered serum triglycerides. LX4211 also mediated trends for lower weight, lower blood pressure, and higher glucagon-like peptide-1 levels. In a follow-up single-dose study in 12 patients with T2DM, LX4211 (300 mg) significantly increased glucagon-like peptide-1 and peptide YY levels relative to pretreatment values, probably by delaying SGLT1-mediated intestinal glucose absorption. In both studies, LX4211 was well tolerated without evidence of increased gastrointestinal side effects. These data support further study of LX4211-mediated dual SGLT1/SGLT2 inhibition as a novel mechanism of action in the treatment of T2DM.

Tumor-associated macrophages (TAMs) are critical drivers of tumor progression and immunosuppression within the tumor microenvironment. The dominant TAM phenotype is broadly characterized as harboring M2-like macrophage properties, which are anti-inflammatory and pro-tumor, as opposed to M1-like macrophages, which possess tumoricidal and pro-inflammatory characteristics. The dependence of M2 TAMs on CSF1 receptor (CSF1R) kinase signaling has made CSF1R a desirable therapeutic target, and the need for highly selective therapies for use in combinations. Through an extensive medicinal chemistry campaign, we identified a series of orally bioavailable, highly potent, exquisitely selective inhibitors of CSF1R (IC50 < 10 nM) with excellent pharmacologic properties that are appropriate for evaluation as a cancer therapy. The aim of our study was to assess the biological impact of our lead CSF1R inhibitor (CSF1Ri) on macrophage populations and the consequent effect on T effector cells. In vitro biochemical activity was evaluated in various kinase assays comparing our CSF1Ri to BLZ945. The compound was evaluated using syngeneic murine models of colorectal cancer (MC38) and pancreatic adenocarcinoma (PANC02). Tumors were immune profiled using NanoString, immunohistochemistry and flow cytometric analysis establishing that there was a depletion of macrophages and a reduction in the relative amount of M2+ (CD206+MHCII-) cells, with a concomitant increase in M1+ (MHCII+CD206-) cells. Myeloid-derived suppressor cells (MDSCs), CD4, and CD8 cell infiltration were also altered with an elevation of cytotoxic immune cells. In conclusion, we have identified and characterized a novel potent and selective inhibitor of CSF1R with highly favorable PK/PD properties, that compares favorably to BLZ945. We also have clear evidence that our novel CSF1R inhibitors modulates TAM infiltration and phenotype altering the immune cell milieu toward a more favorable anti-tumor environment. Citation Format: Erika Suzuki, Jeffrey J. Kovacs, Nakia D. Spencer, Sonal Sonal, Ningping Feng, Angela L. Harris, Robert A. Mullinax, Andy M. Zuniga, Sarah B. Johnson, Mikhila Mahendra, Tin Oo Khor, Faika Mseeh, Zhen Liu, Jason P. Burke, Keith Mikule, Martin Tremblay, Timothy P. Heffernan, Philip Jones, Barbara Czako, Joseph R. Marszalek. Promoting an anti-tumor immune environment with a novel, exquisitely selective CSF1R inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-124.

Indoleamine 2,3-dioxygenase (IDO1 and IDO2) and tryptophan dioxygenase (TDO) are heme-containing enzymes that mediate the rate limiting step in the oxidative degradation of L-tryptophan (L-TRP) to kynurenine (KYN) metabolites. Tryptophan catabolism through the KYN metabolic pathway is now recognized as one of many mechanisms involved in tumor cell evasion of the immune surveillance system. Inhibition of the KYN pathway in the tumor microenvironment can lead to improved immune response and tumor growth suppression. Recently, clinical proof of concept of this mechanism has been demonstrated using an Indoleamine 2,3-dioxygenase (IDO1) inhibitor in combination with a PD-1 antagonist in a variety of tumor contexts. Consideration of known low molecular weight heme-co-ordinating ligands identified from the PDB, in conjunction with a virtual screen performed in-silico identified a number of potentially interesting starting points for medicinal chemistry development. Identification of an attractive indazole fragment as a starting point, and expansion into alternative bicyclic cores, resulted in the discovery of a family of imidazopyridines as potent human IDO1 inhibitors with >200 fold selectivity against TDO. Utilizing a structure-based design approach allowed rapid lead optimization that resulted in the identification of IACS-8968. Crystallography studies were conducted, and binding of IACS-8968 to the heme domain of the human IDO1 was confirmed. The homochiral imidazopyridine IACS-8968 displayed cellular IC50= 29 nM in a HeLa cell line expressing human IDO1 and IC50= 21 nM in a PANC02 mouse cell line expressing the murine IDO1 enzyme, showed satisfactory selectivity margin (> 150 fold) versus its CYP450 inhibition profile and good oral bioavailability across species. PK/PD experiments indicated that, at equivalent exposure, IACS-8968 (sodium salt) and epacadostat decreased tumor KYN at comparable levels in CT26 syngeneic mouse model. Citation Format: Alessia Petrocchi, Naphtali J. Reyna, Faika Mseeh, Connor A. Parker, Simon Yu, Quanyun Xu, Ningping Feng, Paul Leonard, Norma Rogers, Jason B. Cross, Angela L. Harris, Yongying Jiang, Tin Oo Khor, Mikhila G. Mahendra, Jihai Pang, Qi Wu, Andy M. Zuniga, Timothy McAfoos, Timothy McAfoos, Matthew M. Hamilton, Joe R. Marszalek, Keith Mikule, Paul Vancutsem, Keith Wilcoxen, Martin Tremblay, Philip Jones, Richard T. Lewis. Discovery of an imidazopyridine series of potent human IDO1 inhibitors with robust target engagement in a preclinical tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-071.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of oral anti-diabetic agents that improve glycemic control by inhibiting SGLT2-mediated renal glucose reabsorption. Currently available agents increase urinary glucose excretion (UGE) to 50% of filtered glucose when SGLT2 is completely inhibited. This led us to test whether LP-925219, a small molecule dual SGLT1/SGLT2 inhibitor, increases UGE to maximal values in wild-type (WT) mice. We first tested LP-925219 inhibition of glucose transport by HEK293 cells expressing SGLT1 or SGLT2, and then characterized LP-925219 pharmacokinetics. We found that LP-925219 was a potent inhibitor of mouse SGLT1 (IC50 = 22.6 nmol/L) and SGLT2 (IC50 = 0.5 nmol/L), and that a 10 mg/kg oral dose was bioavailable (87%) with a long half-life (7 h). We next delivered LP-925219 by oral gavage to WT, SGLT1 knockout (KO), SGLT2 KO, and SGLT1/SGLT2 double KO (DKO) mice and measured their 24-h UGE. We found that, in vehicle-treated mice, DKO UGE was maximal and SGLT2 KO, SGLT1 KO, and WT UGEs were 30%, 2%, and 0.2% of maximal, respectively; we also found that LP-925219 dosed at 60 mg/kg twice daily increased UGE of SGLT1 KO, SGLT2 KO, and WT mice to DKO UGE levels. These findings show that orally available dual SGLT1/SGLT2 inhibitors can maximize 24-h UGE in mammals, and suggest that such agents merit further evaluation for their potential, in diabetic patients, to achieve better glycemic control than is achieved using selective SGLT2 inhibitors.

Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.

Riboflavin kinase (RFK) is an essential enzyme catalyzing the phosphorylation of riboflavin (vitamin B 2 ) to form FMN, an obligatory step in vitamin B 2 utilization and flavin cofactor synthesis. The structure of human RFK revealed a six-stranded antiparallel β barrel core structurally similar to the riboflavin synthase/ferredoxin reductase FAD binding domain fold. The binding site of an intrinsically bound MgADP defines a novel nucleotide binding motif that encompasses a loop, a 3 10 helix, and a reverse turn followed by a short β strand. This active site loop forms an arch with ATP and riboflavin binding at the opposite side and the phosphoryl transfer appears to occur through the hole underneath the arch. The invariant residues Asn36 and Glu86 are implicated in the catalysis.

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability of E. coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E. coli genes nadD , coaD , and ribF ), are discussed in detail.

Cancer cells display metabolic properties distinct from normal cells and are therefore believed to be dependent on key metabolic enzymes. The effectiveness of targeting metabolism for cancer therapy has been largely restricted due to metabolic adaptability. We postulate that tumors with metabolic vulnerabilities will be limited in such adaptation, thus providing a unique opportunity for therapeutic intervention. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is a metabolically vulnerable cancer, due to mutations in a key TCA cycle enzyme - fumarate hydratase (FH), thus rendering them deficient in oxidative phosphorylation (OXPHOS). We performed a loss-of-function genetic screen in a FH-null HLRCC model (UOK262 cells) and identified 6-phosphogluconate dehydrogenase (6PGD) - a rate-limiting enzyme in the non-oxidative arm of pentose phosphate pathway - as a critical regulator of cell growth. Inhibition of 6PGD using either dox-inducible shRNA or a small molecule inhibitor dramatically blocks the growth of UOK262 cells, and in contrast, has no effect on 6PGD-deficient NB1 cells. The growth inhibition from knockdown is fully rescued by expressing shRNA-resistant 6PGD. Conversely, ectopic expression of 6PGD in NB1 cells results in a gain of function phenotype. Importantly, in vivo, 6PGD knockdown causes robust regression of established UOK262 xenografts. Mechanistically, 6PGD inhibition causes the accumulation of 6-phosphogluconate, blocks glycolysis, and induces ROS production. Furthermore, in tumors with intact TCA cycle, 6PGD inhibition shows synergism with OXPHOS Complex I inhibitor IACS-10759. Together, our data suggest that 6PGD is a bona fide oncology target and provide a strong rationale for developing small molecule inhibitors that could be used to treat OXPHOS-deficient tumors as single agent, or combined with inhibitors of OXPHOS. Citation Format: Yuting Sun, Madhavi Bandi, Timothy Lofton, Melinda Smith, Christopher Bristow, Norma Rogers, Chang Edward, Mary Geck Do, Yongying Jiang, Pietro Morlacchi, Florian Muller, Faika Mseeh, Barbara Czako, Wylie Palmer, Carlo Toniatti, Philip Jones, Giulio Draetta, Timothy Heffernan, Joseph Marszalek. Functional genomics reveals dependency on 6-phosphogluconate dehydrogenase in OXPHOS-deficient tumors. [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 1023.

Sodium-glucose cotransporter 2 (SGLT2) is the major, and SGLT1 the minor, transporter responsible for renal glucose reabsorption. Increasing urinary glucose excretion (UGE) by selectively inhibiting SGLT2 improves glycemic control in diabetic patients. We generated Sglt1 and Sglt2 knockout (KO) mice, Sglt1/Sglt2 double-KO (DKO) mice, and wild-type (WT) littermates to study their relative glycemic control and to determine contributions of SGLT1 and SGLT2 to UGE. Relative to WTs, Sglt2 KOs had improved oral glucose tolerance and were resistant to streptozotocin-induced diabetes. Sglt1 KOs fed glucose-free high-fat diet (G-free HFD) had improved oral glucose tolerance accompanied by delayed intestinal glucose absorption and increased circulating glucagon-like peptide-1 (GLP-1), but had normal intraperitoneal glucose tolerance. On G-free HFD, Sglt2 KOs had 30%, Sglt1 KOs 2%, and WTs

The human skeleton is affected by mutations in Low-density lipoprotein Receptor-related Protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with inducible Lrp5 mutations that cause high bone mass and low bone mass phenotypes in humans. We conditionally-induced Lrp5 mutations in osteocytes and found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also conditionally-induced an Lrp5 mutation in cells that contribute to the appendicular skeleton, and not to the axial skeleton, and we observed bone properties were altered in the limbs, and not in the spine. These data indicate that Lrp5 signaling functions locally and suggest increasing LRP5 signaling in mature bone cells as a strategy to treat human low bone mass disorders, such as osteoporosis.

MTH1 is a protein that sanitizes oxidized dNTPs in the cell. It preferentially hydrolyzes 8-oxo-dGTP and 2-OH-dATP to their corresponding monophosphates and thereby prevents the incorporation of oxidized nucleotides into DNA or RNA. The functional result is a reduction in down-stream mutations, or DNA damage, thus preventing cell death. Recent publications suggest that MTH1 is a non-essential enzyme in normal cells, but is required for the survival of cancer cells as a consequence of being subjected to high levels of oxidative stress; hence its relevance as an oncology target. Gad et al., 2014 The interesting target rationale combined with a perceived high chemical tractability of the target resulting from availability of x-ray crystallographic information for the protein, led to a decision to undertake, in parallel, target validation, assay development and drug discovery. This effort resulted in the structure-based design of potent, cell active MTH1 small molecule inhibitors. This poster will describe the discovery and optimization of these inhibitors, and their use to evaluate the potential of MTH1 as an oncology target. This work was complemented by parallel genetic studies. Pharmacodynamic evaluation of target engagement using proximal biomarkers will be presented, as will phenotypic responses across a range of cancer cell lines. Citation Format: Elisabetta Leo, Alessia Petrocchi, Jennifer Bardenhagen, Maria Alimova, Xi Shi, Connor Parker, Naphtali Reyna, Matthew Hamilton, Edward Felix, Andrzej Mazan, Christian Dillon, Faika Mseeh, Joseph R. Marszalek, Carlo Toniatti, Giulio Draetta, Phil Jones, Richard T. Lewis. Identification of potent, cell active MTH1 inhibitors and their use in target validation studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B48.