[Current Status and Regulatory Issues of Companion Diagnostics in Japan].

Companion diagnostics(CDx)are in vitro diagnostic products that are used to predict the efficacy and adverse effects of therapeutic drugs prior to administration, and are co-developed and co-approved with the therapeutic drugs in principle. In Japan, 40 CDx products have been approved by January 2024, and 39 products are used to determine if therapeutic drugs are applicable for cancer treatment. In the CDx products for cancer treatment, PCR, immunohistochemistry, or in situ hybridization is used to clarify the mutations(point mutations, insertions/deletions, fusions, etc.

Development of Gilteritinib-Based Chimeric Small Molecules that Potently Induce Degradation of FLT3-ITD Protein.

Internal tandem duplication (ITD) in the gene encoding FMS-like tyrosine kinase 3 () (FLT3-ITD) is the most frequently observed mutation in acute myeloid leukemia (AML). Currently approved FLT3 kinase inhibitors have high efficacy, but drug resistance caused by reactivation of FLT3 kinase activity is often clinically observed. In this study, we developed novel FLT3 degraders by introducing gilteritinib, an FDA-approved FLT3 inhibitor, into targeted protein degradation technology.

Development of a potent small-molecule degrader against oncogenic BRAF protein that evades paradoxical MAPK activation.

BRAF mutations are frequently observed in melanoma and hairy-cell leukemia. Currently approved rapidly accelerated fibrosarcoma (RAF) kinase inhibitors targeting oncogenic BRAF V600 mutations have shown remarkable efficacy in the clinic, but their therapeutic benefits are occasionally hampered by acquired resistance due to RAF dimerization-dependent reactivation of the downstream MAPK pathway, which is known as paradoxical activation. There is also a concern that paradoxical activation of the MAPK pathway may trigger secondary cancer progression.

Protocols for Synthesis of SNIPERs and the Methods to Evaluate the Anticancer Effects.

Inducing degradation of undruggable target proteins by the use of chimeric small molecules, represented by proteolysis-targeting chimeras, is a promising strategy for drug development. We developed a series of chimeric molecules, termed "specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers" (SNIPERs) that recruit IAP ubiquitin ligases to induce degradation of target proteins. SNIPERs also induce degradation of some IAPs, including cIAP1 and XIAP, which are antiapoptotic proteins that are overexpressed in many cancers.

Influence of EGFR-activating mutations on sensitivity to tyrosine kinase inhibitors in a KRAS mutant non-small cell lung cancer cell line.

In non-small cell lung cancer (NSCLC), oncogenic driver mutations including those in KRAS and EGFR are typically mutually exclusive. However, recent reports indicate that multiple driver mutations are found in a certain percentage of cancers, and that the therapeutic responses of such cases with co-mutations of driver genes are largely unclear. Here, using CRISPR-Cas9-mediated genome editing, we generated isogenic cell lines harboring one or two copies of an EGFR-activating mutation from the human NSCLC cell line A549, which is known to harbor a homozygous KRAS gene mutation.

Preparation of the standard cell lines for reference mutations in cancer gene-panels by genome editing in HEK 293 T/17 cells.

Background: Next Generation Sequencer (NGS) is a powerful tool for a high-throughput sequencing of human genome. It is important to ensure reliability and sensitivity of the sequence data for a clinical use of the NGS. Various cancer-related gene panels such as Oncomine™ or NCC OncoPanel have been developed and used for clinical studies.

Targeted Protein Degradation by Chimeric Small Molecules, PROTACs and SNIPERs.

Technologies that induce targeted protein degradation by small molecules have been developed recently. Chimeric small molecules such as Proteolysis Targeting Chimeras (PROTACs) and Specific and Non-genetic IAP-dependent Protein Erasers (SNIPERs), and E3 modulators such as thalidomides, hijack the cellular machinery for ubiquitylation, and the ubiquitylated proteins are subjected to proteasomal degradation. This has motivated drug development in industry and academia because "undruggable targets" can now be degraded by targeted protein degradation.

eIF4A inhibition circumvents uncontrolled DNA replication mediated by 4E-BP1 loss in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) relies on hyperactivated protein synthesis. Consistently, human and mouse PDAC lose expression of the translational repressor and mTOR target 4E-BP1. Using genome-wide polysome profiling, we here explore mRNAs whose translational efficiencies depend on the mTOR/4E-BP1 axis in pancreatic cancer cells.

Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2.

Translational control of gene expression plays a key role during the early phases of embryonic development. Here we describe a transcriptional regulator of mouse embryonic stem cells (mESCs), Yin-yang 2 (YY2), that is controlled by the translation inhibitors, Eukaryotic initiation factor 4E-binding proteins (4E-BPs). YY2 plays a critical role in regulating mESC functions through control of key pluripotency factors, including Octamer-binding protein 4 (Oct4) and Estrogen-related receptor-β (Esrrb).

Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3.

Targeting mTORC1 is a highly promising strategy in cancer therapy. Suppression of mTORC1 activity leads to rapid dephosphorylation of eIF4E-binding proteins (4E-BP1-3) and subsequent inhibition of mRNA translation. However, how the different 4E-BPs affect translation during prolonged use of mTOR inhibitors is not known.

TBL2 Associates With ATF4 mRNA Via Its WD40 Domain and Regulates Its Translation During ER Stress.

PKR-like ER-resident kinase (PERK) phosphorylates eukaryotic translation initiation factor 2 α (eIF2α) under endoplasmic reticulum (ER) stress; this results in repression of general translation and induction of specific gene expression, such as activating transcription factor 4 (ATF4). We previously showed that, upon ER stress, transducin (β)-like 2 (TBL2) was an ER-localized transmembrane protein and interacted with PERK and that TBL2 was involved in ATF4 expression and cell survival. Here, we show that TBL2 is able to associate with ATF4 mRNA and regulate its translation.

The endoplasmic reticulum-localized protein TBL2 interacts with the 60S ribosomal subunit.

Transducin (beta)-like 2 (TBL2) is a poorly characterized protein comprising the N-terminal transmembrane region and the C-terminal WD40 domain. We previously showed that TBL2 is an endoplasmic reticulum (ER)-localized protein that interacts with PKR-like ER-resident kinase (PERK), and under ER stress, it mediates protein expression of activating transcription factor 4 (ATF4). However, further molecular characterization of TBL2 is useful to better understand the function of this molecule.

Light-regulated translational control of circadian behavior by eIF4E phosphorylation.

The circadian (∼24 h) clock is continuously entrained (reset) by ambient light so that endogenous rhythms are synchronized with daily changes in the environment. Light-induced gene expression is thought to be the molecular mechanism underlying clock entrainment. mRNA translation is a key step of gene expression, but the manner in which clock entrainment is controlled at the level of mRNA translation is not well understood.

TBL2 is a novel PERK-binding protein that modulates stress-signaling and cell survival during endoplasmic reticulum stress.

Under ER stress, PKR-like ER-resident kinase (PERK) phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4). This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood.

Hyperactivation of 4E-binding protein 1 as a mediator of biguanide-induced cytotoxicity during glucose deprivation.

Biguanides, including metformin, buformin, and phenformin, are potential antitumorigenic agents and induce cell death during glucose deprivation, a cell condition that occurs in the tumor microenvironment. Here, we show that this selective killing of glucose-deprived cells is coupled with hyperactivation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation initiation. We found, in fact, that the 4E-BP1 hyperactivation led to failure of the unfolded protein response (UPR), an endoplasmic reticulum-originated stress signaling pathway for cell survival.

Mitochondria regulate the unfolded protein response leading to cancer cell survival under glucose deprivation conditions.

Cancer cells consume large amounts of glucose because of their specific metabolic pathway. However, cancer cells exist in tumor tissue where glucose is insufficient. To survive, cancer cells likely have the mechanism to elude their glucose addiction.

A novel endoplasmic reticulum export signal: proline at the +2-position from the signal peptide cleavage site.

NUCB1 (nucleobindin 1) is a Golgi-localized soluble protein with a signal peptide and multiple functional domains. We reported recently that NUCB1 is a negative regulator of the unfolded protein response that activates various endoplasmic reticulum (ER)-originating signaling pathways. In that report, we also showed that Golgi localization of NUCB1 was essential to regulate the unfolded protein response.

Caspase-8 mediates mitochondrial release of pro-apoptotic proteins in a manner independent of its proteolytic activity in apoptosis induced by the protein synthesis inhibitor acetoxycycloheximide in human leukemia Jurkat cells.

The cysteine protease caspase-8 plays an essential role in apoptosis induced by death receptors. The protein synthesis inhibitor acetoxycycloheximide (Ac-CHX) has been previously shown to induce rapid apoptosis mediated by the release of cytochrome c in human leukemia Jurkat cells. In this study, the novel molecular mechanism that links caspase-8 to the mitochondrial release of pro-apoptotic proteins has been identified.

Preventing the unfolded protein response via aberrant activation of 4E-binding protein 1 by versipelostatin.

We recently isolated a macrocyclic compound, versipelostatin (VST), that exerts in vivo antitumor activity. VST shows unique, selective cytotoxicity to glucose-deprived tumor cells by preventing the unfolded protein response (UPR). Here we show that eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of eukaryotic initiation factor 4E-mediated protein translation, plays a role in the UPR-inhibitory action of VST.

ERK and p38 MAP kinase are involved in downregulation of cell surface TNF receptor 1 induced by acetoxycycloheximide.

Tumor necrosis factor (TNF)-alpha activates the nuclear factor kappaB (NF-kappaB) signaling pathway. The protein synthesis inhibitor cycloheximide (CHX) and its structural derivative acetoxycycloheximide (Ac-CHX) have been recently shown to block the TNF-alpha-induced activation of NF-kappaB via ectodomain shedding of TNF receptor 1 (TNF-R1) in human lung carcinoma A549 cells. In this study, we show that ERK and p38 MAP kinase are involved in the downregulation of cell surface TNF-R1 upon exposure to Ac-CHX and the subsequent inhibition of TNF-alpha-induced NF-kappaB activation.

Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-alpha-mediated activation of NF-kappaB and caspase-8.

The transcription factor nuclear factor kappaB (NF-kappaB) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-alpha-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-alpha-mediated activation of NF-kappaB and caspase-8 in human lung carcinoma A549 cells.

Etoposide-resistant HT-29 human colon carcinoma cells during glucose deprivation are sensitive to piericidin A, a GRP78 down-regulator.

Glucose deprivation, a pathophysiological cell condition, causes up-regulation of GRP78 and induction of etoposide resistance in human cancer cells. The induction of drug resistance can be partly explained by the fact that GRP78 can block activation of caspase-7 induced by treatment with etoposide. Therefore, downregulating GRP78 expression may be a novel strategy anticancer drug development.

Nucleobindin 1 controls the unfolded protein response by inhibiting ATF6 activation.

In response to endoplasmic reticulum (ER) stress, activating transcription factor 6 (ATF6), an ER membrane-anchored transcription factor, is transported to the Golgi apparatus and cleaved by site-1 protease (S1P) to activate the unfolded protein response (UPR). Here, we identified nucleobindin 1 (NUCB1) as a novel repressor of the S1P-mediated ATF6 activation. NUCB1 is an ER stress-inducible gene with the promoter region having functional cis-elements for transcriptional activation by ATF6.