Antibody to CD137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.

YES1 Is a Targetable Oncogene in Cancers Harboring YES1 Gene Amplification.

Targeting genetic alterations of oncogenes by molecular-targeted agents (MTA) is an effective approach for treating cancer. However, there are still no clinical MTA options for many cancers, including esophageal cancer. We used a short hairpin RNA library to screen for a new oncogene in the esophageal cancer cell line KYSE70 and identified YES proto-oncogene 1 (YES1) as having a significant impact on tumor growth. An analysis of clinical samples showed that YES1 gene amplification existed not only in esophageal cancer but also in lung, head and neck, bladder, and other cancers, indicating that YES1 would be an attractive target for a cancer drug. Because there is no effective YES1 inhibitor so far, we generated a YES1 kinase inhibitor, CH6953755. YES1 kinase inhibition by CH6953755 led to antitumor activity against YES1-amplified cancers in vitro and in vivo. Yes-associated protein 1 (YAP1) played a role downstream of YES1 and contributed to the growth of YES1-amplified cancers. YES1 regulated YAP1 transcription activity by controlling its nuclear translocation and serine phosphorylation. These findings indicate that the regulation of YAP1 by YES1 plays an important role in YES1-amplified cancers and that CH6953755 has therapeutic potential in such cancers. SIGNIFICANCE: These findings identify the SRC family kinase YES1 as a targetable oncogene in esophageal cancer and describe a new inhibitor for YES1 that has potential for clinical utility.See related commentary by Rai, p. 5702.

Acquired JHDM1D-BRAF Fusion Confers Resistance to FGFR Inhibition in FGFR2-Amplified Gastric Cancer.

FGFR2 gene is frequently amplified in gastric cancer. Recently, targeting FGFR2 has drawn attention as a form of gastric cancer therapy, and FGFR-selective inhibitors have shown promising efficacy in clinical studies. Because overcoming acquired resistance is a common problem with molecular targeting drugs, we investigated a resistant mechanism of FGFR inhibitors using the gastric cancer cell line SNU-16, which harbors FGFR2 amplification. We established single-cell clones of FGFR inhibitor-resistant SNU-16 (AZD-R) by continuous exposure to AZD4547, a selective FGFR inhibitor. To screen the genetic alterations acquired in AZD-R, we ran a comparative genomic hybridization assay and found an amplification of Chr7q34 region. The chromosomal breakpoints were located between the 12th and the 13th exon of jumonji C domain containing histone demethylase 1 homolog D (JHDM1D) and between the 3rd and the 4th exon of BRAF We sequenced cDNA of the AZD-R clones and found fusion kinase JHDM1D-BRAF, which has previously been identified in primary ovarian cancer. Because JHDM1D-BRAF fusion lacks a RAS-binding domain, the dimerization of JHDM1D-BRAF was enhanced. A cell growth inhibition assay using MEK inhibitors and RAF-dimer inhibitors indicated the dependence of AZD-R clones for growth on the MAPK pathway. Our data provide a clinical rationale for using a MEK or RAF dimer inhibitor to treat FGFR2-amplified gastric cancer patients who have acquired resistance through the JHDN1D-BRAF fusion. Mol Cancer Ther; 17(10); 2217-25. ©2018 AACR.

Discovery of [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-(1H-indol-2-yl)methanone (CH5183284/Debio 1347), An Orally Available and Selective Fibroblast Growth Factor Receptor (FGFR) Inhibitor.

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases regulates multiple biological processes, such as cell proliferation, migration, apoptosis, and differentiation. Various genetic alterations that drive activation of the receptors and the pathway are associated with tumor growth and survival; therefore, the FGFR family represents an attractive therapeutic target for treating cancer. Here, we report the discovery and the pharmacological profiles of 8 (CH5183284/Debio 1347), an orally available and selective inhibitor of FGFR1, FGFR2, and FGFR3. The chemical modifications, which were guided by 3D-modeling analyses of the inhibitor and FGFRs, led to identifying an inhibitor that is selective to FGFR1, FGFR2, and FGFR3. In in vitro studies and xenograft models in mice, 8 shows antitumor activity against cancer cell lines that harbor genetically altered FGFRs. These results support the potential therapeutic use of 8 as a new anticancer agent.

Activating Mutations in PIK3CB Confer Resistance to PI3K Inhibition and Define a Novel Oncogenic Role for p110ß.

Activation of the PI3K pathway occurs commonly in a wide variety of cancers. Experience with other successful targeted agents suggests that clinical resistance is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN-deficient cancers. We generated cell lines resistant to the pan-PI3K inhibitor GDC-0941 from parental PTEN-null breast cancer cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines that was recurrent in cancer patients. Stable expression of mutant PIK3CB variants conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. Furthermore, we show that the p110ß D1067Y mutant was highly activated and induced PIP3 levels at the cell membrane, subsequently promoting the localization and activation of AKT and PDK1 at the membrane and driving PI3K signaling to a level that could withstand treatment with proximal inhibitors. Finally, we demonstrate that the PIK3CB D1067Y mutant behaved as an oncogene and transformed normal cells, an activity that was enhanced by PTEN depletion. Collectively, these novel preclinical and clinical findings implicate the acquisition of activating PIK3CB D1067 mutations as an important event underlying the resistance of cancer cells to selective PI3K inhibitors.

ERK Signal Suppression and Sensitivity to CH5183284/Debio 1347, a Selective FGFR Inhibitor.

Drugs that target specific gene alterations have proven beneficial in the treatment of cancer. Because cancer cells have multiple resistance mechanisms, it is important to understand the downstream pathways of the target genes and monitor the pharmacodynamic markers associated with therapeutic efficacy. We performed a transcriptome analysis to characterize the response of various cancer cell lines to a selective fibroblast growth factor receptor (FGFR) inhibitor (CH5183284/Debio 1347), a mitogen-activated protein kinase kinase (MEK) inhibitor, or a phosphoinositide 3-kinase (PI3K) inhibitor. FGFR and MEK inhibition produced similar expression patterns, and the extracellular signal-regulated kinase (ERK) gene signature was altered in several FGFR inhibitor-sensitive cell lines. Consistent with these findings, CH5183284/Debio 1347 suppressed phospho-ERK in every tested FGFR inhibitor-sensitive cell line. Because the mitogen-activated protein kinase (MAPK) pathway functions downstream of FGFR, we searched for a pharmacodynamic marker of FGFR inhibitor efficacy in a collection of cell lines with the ERK signature and identified dual-specificity phosphatase 6 (DUSP6) as a candidate marker. Although a MEK inhibitor suppressed the MAPK pathway, most FGFR inhibitor-sensitive cell lines are insensitive to MEK inhibitors and we found potent feedback activation of several pathways via FGFR. We therefore suggest that FGFR inhibitors exert their effect by suppressing ERK signaling without feedback activation. In addition, DUSP6 may be a pharmacodynamic marker of FGFR inhibitor efficacy in FGFR-addicted cancers.

Mechanism of Oncogenic Signal Activation by the Novel Fusion Kinase FGFR3-BAIAP2L1.

Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding FGFR family members. However, most fusion genes are not functionally characterized, and their potentials in targeted therapy are unclear. We investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). We identified 4 patients with bladder cancer and 2 patients with lung cancer harboring the FGFR3-BAIAP2L1 fusion through PCR and FISH assay screens. To investigate the oncogenic potential of the fusion gene, we established an FGFR3-BAIAP2L1 transfectant with Rat-2 fibroblast cells (Rat-2_F3-B). The FGFR3-BAIAP2L1 fusion had transforming activity in Rat2 cells, and Rat-2_F3-B cells were highly tumorigenic in mice. Rat-2_F3-B cells showed in vitro and in vivo sensitivity in the selective FGFR inhibitor CH5183284/Debio 1347, indicating that FGFR3 kinase activity is critical for tumorigenesis. Gene signature analysis revealed that FGFR3-BAIAP2L1 activates growth signals, such as the MAPK pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. We also established Rat-2_F3-B-ΔBAR cells expressing an FGFR3-BAIAP2L1 variant lacking the Bin-Amphiphysin-Rvs (BAR) dimerization domain of BAIAP2L1, which exhibited decreased tumorigenic activity, FGFR3 phosphorylation, and F3-B-ΔBAR dimerization, compared with Rat-2_F3-B cells. Collectively, these data suggest that constitutive dimerization through the BAR domain promotes constitutive FGFR3 kinase activation and is essential for its potent oncogenic activity.

The fibroblast growth factor receptor genetic status as a potential predictor of the sensitivity to CH5183284/Debio 1347, a novel selective FGFR inhibitor.

The FGF receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic alterations such as gene amplifications, point mutations, or chromosomal translocations/rearrangements. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGF receptor (VEGFR) or platelet-derived growth factor receptor (PDGFR) family displayed clinical benefits in cohorts of patients with FGFR genetic alterations. However, to achieve more potent and prolonged activity in such populations, a selective FGFR inhibitor is still needed. Here, we report the identification of CH5183284/Debio 1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold. By interacting with unique residues in the ATP-binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 but does not inhibit kinase insert domain receptor (KDR) or other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 displayed preferential antitumor activity against cancer cells with various FGFR genetic alterations in a panel of 327 cancer cell lines and in xenograft models. Because of its unique binding mode, CH5183284/Debio 1347 can inhibit FGFR2 harboring one type of the gatekeeper mutation that causes resistance to other FGFR inhibitors and block FGFR2 V564F-driven tumor growth. CH5183284/Debio 1347 is under clinical investigation for the treatment of patients harboring FGFR genetic alterations.

[Changes in life styles of pregnant women and risks for having a pregnancy afflicted with spina bifida].

AIMS: Though periconceptional intakes of folic acid could prevent the occurrence of spina bifida by 50 to 70%, the prevalence has not shown any decreasing tendency during the past 30 years in Japan. We aim to analyze various parameters through life style questionnaires and 3-day food records obtained from pregnant women the last 10 years, and to examine whether their life styles have been shifting to the direction of lowering the incidence of spina bifida. MATERIALS AND METHODS: Life style questionnaires inquired of knowledge of folic acid in relation to preventing spina bifida during a pregnancy and other relevant parameters, which were collected from 11,861 participants during a period of from 2002 to 2011. Food records asked participants to semi-quantitatively describe diets and beverages they consumed for a 3-day period, which were collected from 1,081 pregnant women from 2003 to 2011. RESULTS: Life style questionnaires demonstrated that knowledge of folic acid and the proportion of those who took folic acid supplements elevated from 15.3 and 9.1% in 2002 to 43.7 and 61.5% in 2011, respectively, that comparison of those who took folic acid supplements from 2008 to 2011 residing in one of 8 districts of Japan showed a significant difference, i.e., the proportion of those in the Chugoku or Kyushu district being significantly lower compared to that in the Hokkaido district, and that other life style parameters have not much altered the past 9 years, e.g., those who conceived as planed being 67%, those who confirmed own pregnancy within 6 weeks of pregnancy being 70%, those who took balanced diets being 65%, and those who did not smoke or drink being 95% and 96%, respectively. Three-day food records revealed that the mean dietary folate intakes ranged from 260 to 360 microg/day in each year which were less than the recommended dietary allowance (RDA) publicized by the government, but that the proportion of pregnant women in the first trimester who consumed folic acid supplements from 4 weeks prior to to 12 weeks after conception increased from 7.4% in 2003 to 69.6% in 2011. CONCLUSIONS: As a whole it could be stated that life styles of pregnant women have been shifting toward the direction the past 10 years where the risk for having a pregnancy afflicted with spina bifida is to be decreased. Medical doctors, nurses, midwives, dietitians and pharmacists are asked to repeatedly supply important information on folic acid and to advise taking folic acid supplements 400 microg a day to women planning to conceive or women in the reproductive age.

Preclinical antitumor activity of the novel heat shock protein 90 inhibitor CH5164840 against human epidermal growth factor receptor 2 (HER2)-overexpressing cancers.

Heat shock protein 90 (Hsp90), a molecular chaperone that plays a significant role in the stability and maturation of client proteins, including oncogenic targets for cell transformation, proliferation, and survival, is an attractive target for cancer therapy. We identified the novel Hsp90 inhibitor, CH5164840, and investigated its induction of oncogenic client protein degradation, antiproliferative activity, and apoptosis against an NCI-N87 gastric cancer cell line and a BT-474 breast cancer cell line. Interestingly, CH5164840 demonstrated tumor selectivity both in vitro and in vivo, binding to tumor Hsp90 (which forms active multiple chaperone complexes) in vitro, and being distributed effectively to tumors in a mouse model, which, taken together, supports the decreased levels of phosphorylated Akt by CH5164840 that we observed in tumor tissues, but not in normal tissues. As well as being well tolerated, the oral administration of CH5164840 exhibited potent antitumor efficacy with regression in NCI-N87 and BT-474 tumor xenograft models. In addition, CH5164840 significantly enhanced antitumor efficacy against gastric and breast cancer models when combined with the human epidermal growth factor receptor 2 (HER2)-targeted agents, trastuzumab and lapatinib. These data demonstrate the potent antitumor efficacy of CH5164840 when administered alone, and its significant combination efficacy when combined with trastuzumab or lapatinib, supporting the clinical development of CH5164840 as an Hsp90 inhibitor for combination therapy with HER2-targeted agents against HER2-overexpressing tumors.

Functional analysis and subcellular location of two flavohemoglobins from Aspergillus oryzae.

Multiple flavohemoglobin (FHb) homolog genes are found in the genomes of eukaryotic microorganisms, but their functions remain unknown. In this study, two distinct types of FHbs (predictive cytosolic FHb1 and predictive mitochondrial FHb2) from the fungus Aspergillus oryzae were investigated to elucidate the physiological roles of these FHbs. The fhb1 gene responded to external nitric oxide (NO) stress at the transcriptional level, whereas the fhb2 gene did not. Disrupting fhb1 increased cell hypersensitivity to NO stress, whereas deficiency of the fhb2 gene had no effect on phenotype compared to the wild-type strain. By fusing GFP protein to FHbs, we determined that FHb1 and FHb2 are located in the cytosol and mitochondria, respectively. In the wild-type strain, the transcriptional level of the fhb2 gene was too low to be detected, but its expression was detectable in the NirK (mitochondrial copper-containing dissimilatory nitrite reductase) overexpression strain (AoHnirK), which showed a significantly higher denitrification capability than that shown by the wild-type strain. The induction of the fhb2 gene in the AoHnirK strain may be due to the abundance of NO produced by overexpressed NirK in the mitochondria. These results suggest that FHb1 and FHb2 may play a role in protecting cells from external and internal NO stress, respectively.

A eukaryotic copper-containing nitrite reductase derived from a NirK homolog gene of Aspergillus oryzae.

We cloned a bacterial copper-containing nitrite reductase (NirK) homolog gene of Aspergillus oryzae (AonirK). Alignment showed that amino acid residues crucial for copper binding are conserved in the deduced sequence of the fungal protein. The recombinant protein exhibited distinct nitrite reductase activity, and its absorption and EPR spectra showed the presence of type 1 and type 2 copper atoms in the molecule. AonirK transcriptionally responded to denitrification conditions. Although the denitrifying activity of A. oryzae was weak under the conditions employed, high expression of the gene in the fungal cells enhanced the denitrifying activity 6-fold, accompanied by distinct cell growth. Furthermore, the highly expressed AoNirK was subcellularly localized to the mitochondria. The results demonstrated that AoNirK is responsible for fungal denitrification. Discussion is added on the novel insight concerning the origin and evolution of the mitochondrion provided by the findings for eukaryotic NirKs.

Aspergillus oryzae flavohemoglobins promote oxidative damage by hydrogen peroxide.

Apart from their well-established role in nitric oxide detoxification, flavohemoglobins (FHbs) are also believed to be involved in protection against oxidative stress in some yeast and bacteria. However, different studies have reported contradictory results in this regard. Here, we investigate the relationship between two FHbs in Aspergillus oryzae (cytosolic FHb1 and mitochondrial FHb2) and oxidative stress. The strains deficient in the two FHbs exhibited higher resistance to hydrogen peroxide than the wild-type. In addition, the FHb2 overexpression strain showed hypersensitivity to hydrogen peroxide. Flavin reductase accompanied by the ferric reductase activities of the two FHbs were observed in correspondence with this expression. The reductase activities of the FHbs were attributed to their C-terminal flavin reductase domains. The reduced intracellular free iron can subsequently promote oxidative damage by accelerating the Fenton reaction in the cytosol and mitochondria (corresponding to the subcellular localizations of the two FHbs). This study is the first to show that fungal FHbs have a deleterious effect on oxidative protection, and suggests that the accelerated Fenton reaction induced by FHbs might be responsible for this effect.

Distinct enzymatic and cellular characteristics of two secretory phospholipases A2 in the filamentous fungus Aspergillus oryzae.

Microbial secretory phospholipases A(2) (sPLA(2)s) are among the last discovered and least known members of this functionally diverse family of enzymes. We analyzed here two sPLA(2)s, named sPlaA and sPlaB, of the filamentous ascomycete Aspergillus oryzae. sPlaA and sPlaB consist of 222 and 160 amino acids, respectively, and share the conserved Cys and catalytic His-Asp residues typical of microbial sPLA(2)s. Two sPLA(2)s differ in pH optimum, Ca(2+) requirement and expression profile. The splaA mRNA was strongly upregulated in response to carbon starvation, oxidative stress and during conidiation, while splaB was constitutively expressed at low levels and was weakly upregulated by heat shock. Experiments with sPLA(2) overexpressing strains demonstrated that two enzymes produce subtly different phospholipid composition variations and also differ in their subcellular localization: sPlaA is most abundant in hyphal tips and secreted to the medium, whereas sPlaB predominantly localizes to the ER-like intracellular compartment. Both sPLA(2) overexpressing strains were defective in conidiation, which was more pronounced for sPlaB overexpressors. Although no major morphological abnormality was detected in either DeltasplaA or DeltasplaB mutants, hyphal growth of DeltasplaB, but not that of DeltasplaA, displayed increased sensitivity to H(2)O(2) treatment. These data indicate that two A. oryzae sPLA(2) enzymes display distinct, presumably non-redundant, physiological functions.

A signature-based method for indexing cell cycle phase distribution from microarray profiles.

BACKGROUND: The cell cycle machinery interprets oncogenic signals and reflects the biology of cancers. To date, various methods for cell cycle phase estimation such as mitotic index, S phase fraction, and immunohistochemistry have provided valuable information on cancers (e.g. proliferation rate). However, those methods rely on one or few measurements and the scope of the information is limited. There is a need for more systematic cell cycle analysis methods. RESULTS: We developed a signature-based method for indexing cell cycle phase distribution from microarray profiles under consideration of cycling and non-cycling cells. A cell cycle signature masterset, composed of genes which express preferentially in cycling cells and in a cell cycle-regulated manner, was created to index the proportion of cycling cells in the sample. Cell cycle signature subsets, composed of genes whose expressions peak at specific stages of the cell cycle, were also created to index the proportion of cells in the corresponding stages. The method was validated using cell cycle datasets and quiescence-induced cell datasets. Analyses of a mouse tumor model dataset and human breast cancer datasets revealed variations in the proportion of cycling cells. When the influence of non-cycling cells was taken into account, "buried" cell cycle phase distributions were depicted that were oncogenic-event specific in the mouse tumor model dataset and were associated with patients' prognosis in the human breast cancer datasets. CONCLUSION: The signature-based cell cycle analysis method presented in this report, would potentially be of value for cancer characterization and diagnostics.

Cloning and characterization of two flavohemoglobins from Aspergillus oryzae.

Two flavohemoglobin (FHb) genes, fhb1 and fhb2, were cloned from Aspergillus oryzae. The amino acid sequences of the deduced FHb1 and FHb2 showed high identity to other FHbs except for the predicted mitochondrial targeting signal in the N-terminus of FHb2. The recombinant proteins displayed absorption spectra similar to those of other FHbs. FHb1 and FHb2 were estimated to be a monomer and a dimer in solution, respectively. Both of the isozymes exhibit high NO dioxygenase (NOD) activity. FHb1 utilizes either NADH or NADPH as an electron donor, whereas FHb2 can only use NADH. These results suggest that FHb1 and FHb2 are fungal counterparts of bacterial FHbs and act as NO detoxification enzymes in the cytosol and mitochondria, respectively. This study is the first to show that a microorganism contains two isozymes of FHb and that intracellular localization of the isozymes could differ.