Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis.

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

Selective Pharmaceutical Inhibition of PARP14 Mitigates Allergen-Induced IgE and Mucus Overproduction in a Mouse Model of Pulmonary Allergic Response.

The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor α-chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions including asthma. STAT6 binds poly(ADP-ribose) polymerase (PARP)14, an ADP-ribosyl monotransferase. Elimination of PARP14 by gene targeting led to attenuation of OVA-specific allergic lung inflammation. However, PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation, and it is not clear whether inhibition of the catalytic function has any biological consequence. Using BALB/c mice sensitized to the allergen Alternaria alternata, we show that peroral administration of RBN012759, a highly selective inhibitor of ADP-ribosylation by PARP14 with negligible impact on other members of the PARP gene family, achieved biologically active plasma concentrations and altered several responses to the Ag. Specifically, the pharmaceutical compound decreased mucus after allergen challenge, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that PARP14 catalytic activity can contribute to pathogenesis in allergic or atopic processes and propose that other biological endpoints dependent on ADP-ribosylation by PARP14 can be targeted using selective inhibition.

A potent and selective PARP14 inhibitor decreases protumor macrophage gene expression and elicits inflammatory responses in tumor explants.

PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 µM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.

PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition triggers antitumor immunity.

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.

A role for iNOS in fasting hyperglycemia and impaired insulin signaling in the liver of obese diabetic mice.

Chronic inflammation has been postulated to play an important role in the pathogenesis of insulin resistance. Inducible nitric oxide synthase (iNOS) has been implicated in many human diseases associated with inflammation. iNOS deficiency was shown to prevent high-fat diet-induced insulin resistance in skeletal muscle but not in the liver. A role for iNOS in fasting hyperglycemia and hepatic insulin resistance, however, remains to be investigated in obesity-related diabetes. To address this issue, we examined the effects of a specific inhibitor for iNOS, L-NIL, in obese diabetic (ob/ob) mice. iNOS expression was increased in the liver of ob/ob mice compared with wild-type mice. Treatment with iNOS inhibitor reversed fasting hyperglycemia with concomitant amelioration of hyperinsulinemia and improved insulin sensitivity in ob/ob mice. iNOS inhibitor also increased the protein expression of insulin receptor substrate (IRS)-1 and -2 1.5- and 2-fold, respectively, and enhanced IRS-1- and IRS-2-mediated insulin signaling in the liver of ob/ob mice. Exposure to NO donor and ectopically expressed iNOS decreased the protein expression of IRS-1 and -2 in cultured hepatocytes. These results suggest that iNOS plays a role in fasting hyperglycemia and contributes to hepatic insulin resistance in ob/ob mice.

MTAP Deletions in Cancer Create Vulnerability to Targeting of the MAT2A/PRMT5/RIOK1 Axis.

Homozygous 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.

Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3' Splice Site Selection through Use of a Different Branch Point.

Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.

Involvement of the serotonergic system in orexin-induced behavioral alterations in rats.

We have demonstrated involvement of the serotonergic system in orexin-induced behavioral responses in rats. Orexin-A and -B (hypocretin-1 and -2) significantly increased total locomotor activity when administered centrally. They also induced behavioral alterations; increasing grooming, face washing and wet dog shaking in rats. Haloperidol inhibited orexin-induced hyperlocomotion and these behavioral alterations. Serotonin antagonists, ritanserin and metergoline, did not attenuate orexin-induced hyperlocomotion but partly inhibited orexin-induced behavioral alterations. These results suggest that the dopaminergic system might be involved in orexin-induced hyperlocomotion, while both the serotonergic system as well as the dopaminergic system might be involved in orexin-induced behavioral responses.

An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells.

The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy. One potential drug target is isocitrate dehydrogenase 1 (IDH1), which is mutated in multiple human cancers. Here, we examine the role of mutant IDH1 in fully transformed cells with endogenous IDH1 mutations. A selective R132H-IDH1 inhibitor (AGI-5198) identified through a high-throughput screen blocked, in a dose-dependent manner, the ability of the mutant enzyme (mIDH1) to produce R-2-hydroxyglutarate (R-2HG). Under conditions of near-complete R-2HG inhibition, the mIDH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. Blockade of mIDH1 impaired the growth of IDH1-mutant--but not IDH1-wild-type--glioma cells without appreciable changes in genome-wide DNA methylation. These data suggest that mIDH1 may promote glioma growth through mechanisms beyond its well-characterized epigenetic effects.

Evidence of mTOR Activation by an AKT-Independent Mechanism Provides Support for the Combined Treatment of PTEN-Deficient Prostate Tumors with mTOR and AKT Inhibitors.

Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.

Discovery of the First Potent Inhibitors of Mutant IDH1 That Lower Tumor 2-HG in Vivo.

Optimization of a series of R132H IDH1 inhibitors from a high throughput screen led to the first potent molecules that show robust tumor 2-HG inhibition in a xenograft model. Compound 35 shows good potency in the U87 R132H cell based assay and ∼90% tumor 2-HG inhibition in the corresponding mouse xenograft model following BID dosing. The magnitude and duration of tumor 2-HG inhibition correlates with free plasma concentration.

Small molecule activation of PKM2 in cancer cells induces serine auxotrophy.

Proliferating 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.

Discovery of a 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one (MK-2461) inhibitor of c-Met kinase for the treatment of cancer.

c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.

PDK1 attenuation fails to prevent tumor formation in PTEN-deficient transgenic mouse models.

PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target.

MK-2461, a novel multitargeted kinase inhibitor, preferentially inhibits the activated c-Met receptor.

The receptor tyrosine kinase c-Met is an attractive target for therapeutic blockade in cancer. Here, we describe MK-2461, a novel ATP-competitive multitargeted inhibitor of activated c-Met. MK-2461 inhibited in vitro phosphorylation of a peptide substrate recognized by wild-type or oncogenic c-Met kinases (N1100Y, Y1230C, Y1230H, Y1235D, and M1250T) with IC(50) values of 0.4 to 2.5 nmol/L. In contrast, MK-2461 was several hundredfold less potent as an inhibitor of c-Met autophosphorylation at the kinase activation loop. In tumor cells, MK-2461 effectively suppressed constitutive or ligand-induced phosphorylation of the juxtamembrane domain and COOH-terminal docking site of c-Met, and its downstream signaling to the phosphoinositide 3-kinase-AKT and Ras-extracellular signal-regulated kinase pathways, without inhibiting autophosphorylation of the c-Met activation loop. BIAcore studies indicated 6-fold tighter binding to c-Met when it was phosphorylated, suggesting that MK-2461 binds preferentially to activated c-Met. MK-2461 displayed significant inhibitory activities against fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor, and other receptor tyrosine kinases. In cell culture, MK-2461 inhibited hepatocyte growth factor/c-Met-dependent mitogenesis, migration, cell scatter, and tubulogenesis. Seven of 10 MK-2461-sensitive tumor cell lines identified from a large panel harbored genomic amplification of MET or FGFR2. In a murine xenograft model of c-Met-dependent gastric cancer, a well-tolerated oral regimen of MK-2461 administered at 100 mg/kg twice daily effectively suppressed c-Met signaling and tumor growth. Similarly, MK-2461 inhibited the growth of tumors formed by s.c. injection of mouse NIH-3T3 cells expressing oncogenic c-Met mutants. Taken together, our findings support further preclinical development of MK-2461 for cancer therapy.

FGFR2-amplified gastric cancer cell lines require FGFR2 and Erbb3 signaling for growth and survival.

We have identified a critical role for amplified FGFR2 in gastric cancer cell proliferation and survival. In a panel of gastric cancer cell lines, fibroblast growth factor receptor 2 (FGFR2) was overexpressed and tyrosine phosphorylated selectively in FGFR2-amplified cell lines KatoIII, Snu16, and OCUM-2M. FGFR2 kinase inhibition by a specific small-molecule inhibitor resulted in selective and potent growth inhibition in FGFR2-amplified cell lines, resulting in growth arrest in KatoIII cells and prominent induction of apoptosis in both Snu16 and OCUM-2M cells. FGFR2-amplified cell lines also contained elevated phosphotyrosine in EGFR, Her2, and Erbb3, but the elevated phosphorylation in EGFR could not be inhibited by gefitinib or erlotinib. We show that the elevated EGFR, Her2, and Erbb3 phosphotyrosine is dependent on FGFR2, revealing EGFR family kinases to be downstream targets of amplified FGFR2. Moreover, shRNA to Erbb3 resulted in a loss of proliferation, confirming a functional role for the activated EGFR signaling pathway. These results reveal that both the FGFR2 and EGFR family signaling pathways are activated in FGFR2-amplified gastric cancer cell lines to drive cell proliferation and survival. Inhibitors of FGFR2 or Erbb3 signaling may have therapeutic efficacy in the subset of gastric cancers containing FGFR2 amplification.

Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP.

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.