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Aberrant expression of ecotropic viral integration site-1 (EVI1+) is associated with very poor outcomes in acute myeloid leukemia (AML), mechanisms of which are only partially understood. Using the green fluorescent protein reporter system to monitor EVI1 promoter activity, we demonstrated that Evi1high KMT2A-MLLT1–transformed AML cells possess distinct features from Evi1low cells: the potential for aggressive disease independent of stem cell activity and resistance to cytotoxic chemotherapy, along with the consistent gene expression profiles. RNA sequencing and chromatin immunoprecipitation sequencing in EVI1-transformed AML cells and normal hematopoietic cells combined with functional screening by cell proliferation–related short hairpin RNAs revealed that the erythroblast transformation–specific transcription factor ERG (E26 transformation-specific [ETS]-related gene) and cyclin D1 were downstream targets and therapeutic vulnerabilities of EVI1+ AML. Silencing Erg in murine EVI1+ AML models severely impaired cell proliferation, chemoresistance, and leukemogenic capacity. Cyclin D1 is also requisite for efficient EVI1-AML development, associated with gene expression profiles related to chemokine production and interferon signature, and T- and natural killer–cell exhaustion phenotype, depending on the interferon gamma (IFN-γ)/STAT1 pathway but not on CDK4/CDK6. Inhibiting the IFN-γ/STAT1 pathway alleviated immune exhaustion and impaired EVI1-AML development. Overexpression of EVI1 and cyclin D1 was associated with IFN-γ signature and increased expression of chemokines, with increased exhaustion molecules in T cells also in human AML data sets. These data collectively suggest that ERG and cyclin D1 play pivotal roles in the biology of EVI1+ AML, where ERG contributes to aggressive disease nature and chemoresistance, and cyclin D1 leads to IFN-γ signature and exhausted T-cell phenotypes, which could potentially be targeted.

Resistance to immune-checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold-, myeloid cell-, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell-subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC.

Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein–coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell–redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells in vitro and in vivo in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.

Supplementary Tables S1-S2. Supplementary Table S1: Patients' characteristics for flow cytometry analyses on malignant cells from multiple myeloma. Supplementary Table S2: Sequence information of GPA0018 TRAB and GPA0039 TRAB.

Supplementary Table S1 Genetic status information of cancer cell lines Supplementary Table S2 List of probe that is significantly modulated by CH5183284/Debio 1347 Supplementary Table S3 In vivo efficacy data of CH5183284/Debio 1347 Supplementary Figure S1. MAPK pathway suppression by CH5183284/Debio 1347 in FGFR genetically altered cancer cell lines Supplementary Figure S2. Modulation of DUSP6 expression in FGFR inhibitor sensitive cancer cell lines

Supplementary Figures S1-S7. Figure S1: Cell surface expression of GPRC5D on malignant cells from multiple myeloma (patient number: MM#2 to MM#11 in Supplementary Table S1). Figure S2: Cell surface expression of GPRC5D on cancer cell lines and cytotoxicity of GPRC5D TRABs when co-culturing GPRC5D-expressing NCI-H929 cells without PBMC. Figure S3: Cytotoxicity of GPRC5D TRABs when co-culturing GPRC5D-expressing NCI-H929 cells with CD4+ T cells or CD8+ T cells. Figure S4: Antitumor activity and plasma concentrations of GPA0039 TRAB in mouse models with NCI-H929 tumors. Figure S5: T-cell activation of GPRC5D TRABs after co-culturing with GloResponse{trade mark, serif} NFAT-luc2 Jurkat cells and NCI-H929 cells or Human Bone Marrow CD34+ cells (StemCell Technologies) from two different donors. Figure S6: mRNA expression (TPM value) of GPRC5D and BCMA using the GTEx sample set. Figure S7: Binding activity of 100 nM of anti-GPRC5D antibodies on CHO cells expressing cynomolgus monkey GPRC5D.

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Diadumene lineata is a colorful sea anemone with orange stripe tissue of the body column and plain tentacles with red lines. We subjected Diadumene lineata to expression cloning and obtained genes encoding orange (OFP: DiLiFP561) and red fluorescent proteins (RFPs: DiLiFP570 and DiLiFP571). These proteins formed obligatory tetramers. All three proteins showed bright fluorescence with the brightness of 58.3 mM−1·cm−1 (DiLiFP561), 43.9 mM−1·cm−1 (DiLiFP570), and 31.2 mM−1·cm−1 (DiLiFP571), which were equivalent to that of commonly used red fluorescent proteins. Amplitude-weighted average fluorescence lifetimes of DiLiFP561, DiLiFP570 and DiLiFP571 were determined as 3.7, 3.6 and 3.0 ns. We determined a crystal structure of DiLiFP570 at 1.63 Å resolution. The crystal structure of DiLiFP570 revealed that the chromophore has an extended π-conjugated structure similar to that of DsRed. Most of the amino acid residues surrounding the chromophore were common between DiLiFP570 and DiLiFP561, except M159 of DiLiFP570 (Lysine in DiLiFP561), which is located close to the chromophore hydroxyl group. Interestingly, a similar K-to-M substitution has been reported in a red-shifted variant of DsRed (mRFP1). It is a striking observation that the naturally evolved color-change variants are consistent with the mutation induced via protein engineering processes. The newly cloned proteins are promising as orange and red fluorescent markers for imaging with long fluorescence lifetime. Graphical abstract

Inflammatory factors in the peripheral blood, such as the C-reactive protein level and neutrophil-to-lymphocyte ratio (NLR), are prognostic markers in multiple types of cancer, including non-small cell lung cancer (NSCLC). However, the association between inflammatory factors and prognosis based on histological types has not been adequately reported. In addition, the relationship between these factorsand the immune condition of the tumor microenvironment (TME) is unclear. In this study, we first investigated the relationship between preoperative inflammatory markers and clinical outcomes in 176 patients with NSCLC who underwent surgery. Lung adenocarcinoma (LUAD) showed no significant prognostic marker, whereas for lung squamous cell carcinoma (LUSC), a multivariate analysis showed that a high NLR was significantly associated with postoperative recurrence. In LUSC patients, the median time of postoperative recurrence-free survival in patients with a low NLR was longer than that in patients with a high NLR. We then compared the tumor-infiltrating lymphocyte (TIL) profile with inflammatory markers in peripheral blood and found that the NLR was negatively correlated with the frequencies of T cells and B cells in LUSC tissues. Thus, the NLR is a useful predictive biomarker for postoperative recurrence and may reflect the immune condition of the TME in LUSC.

Although JAK1/2 inhibition is effective in alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here, we established induced pluripotent stem cells (iPSCs) derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were used for chemical screening, and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutation recurrently detected in MF patients, were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 γ subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515L decreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes, such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

A 71-year-old female with type B3 thymoma developed severe aplastic anemia. Anti-thymocyte globulin was administered with glucocorticoids and cyclosporin A as the treatment for aplastic anemia. Computed tomography scan revealed that thymoma apparently shrank and remained without regrowth for at least 7 months. As previously reported, glucocorticoid has therapeutic effects on thymoma especially with abundant lymphocytes. Anti-thymocyte globulin also depletes peripheral lymphocytes, but its efficacy in the treatment of thymoma is unknown. Anti-thymocyte globulin and glucocorticoids may have cooperated with each other in reducing thymoma in our case. More cases should be accumulated to elucidate the effects of anti-thymocyte globulin on thymoma.

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson's and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via cell-attached single-channel electrophysiology we show that heteropentamers exhibit currents in between those of K and L variants. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR-α3 channels, which is important to understand and tackle α3 related diseases. ispartof: CELLULAR AND MOLECULAR LIFE SCIENCES vol:79 issue:11 ispartof: location:Switzerland status: published

Autoimmune pancytopenia is rarely seen with Hodgkin lymphoma, and only one pediatric case of pancytopenia after autologous hematopoietic stem cell transplantation (HSCT) has been reported. We herein report a case of autoimmune pancytopenia that developed after autologous HSCT for nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). A 56-year-old Japanese woman underwent autologous HSCT for NLPHL. She developed autoimmune pancytopenia seven months after autologous HSCT. In this case, PSL was effective, and the blood cell counts normalized completely. However, the patient suffered from a fatal infection, probably because of immunosuppression caused by prolonged administration of PSL, as well as a history of several chemotherapies and autologous HSCT. To our knowledge, this is the first adult case of autoimmune pancytopenia after autologous HSCT for Hodgkin lymphoma. To further validate the optimal treatment strategy for autoimmune cytopenia after autologous HSCT, more cases are necessary.

Evi1 is a transcription factor essential for the development as well as progression of acute myeloid leukemia (AML) and high Evi1 AML is associated with extremely poor clinical outcome. Since targeting metabolic vulnerability is the emerging therapeutic strategy of cancer, we herein investigated a novel therapeutic target of Evi1 by analyzing transcriptomic, epigenetic, and metabolomic profiling of mouse high Evi1 leukemia cells. We revealed that Evi1 overexpression and Evi1‐driven leukemic transformation upregulate transcription of gluconeogenesis enzyme Fbp1 and other pentose phosphate enzymes with interaction between Evi1 and the enhancer region of these genes. Metabolome analysis using Evi1‐overexpressing leukemia cells uncovered pentose phosphate pathway upregulation by Evi1 overexpression. Suppression of Fbp1 as well as pentose phosphate pathway enzymes by shRNA‐mediated knockdown selectively decreased Evi1‐driven leukemogenesis in vitro. Moreover, pharmacological or shRNA‐mediated Fbp1 inhibition in secondarily transplanted Evi1‐overexpressing leukemia mouse significantly decreased leukemia cell burden. Collectively, targeting FBP1 is a promising therapeutic strategy of high Evi1 AML., The activated pentose phosphate pathway enhances progression of Evi1‐overexpressing leukemia. Fbp1 and other glucose metabolism‐related genes cooperatively upregulate pentose phosphate pathway activity.

Bioluminescence resonance energy transfer (BRET) has gained impetus to monitor protein interactions in proximity. BRET involves the energy transfer from a bioluminescent donor (luciferases) to a fluorescent acceptor. Since bioluminescence is an intrinsic phenomenon, BRET excludes the need for external illumination and serves as a powerful alternative to fluorescence-based systems. However, BRET has not been widely adopted for single-cell imaging applications, mainly due to the low signal output resulting in poor signal-to-noise ratio. In this chapter, we describe a protocol to optimize spatiotemporal BRET imaging by adopting fluorescent HaloTag acceptors, adapting cell culture conditions and microscopic setup.

Förster resonance energy transfer (FRET) between fluorescent proteins has become a common platform for designing genetically encoded biosensors. For live cell imaging, the acceptor-to-donor intensity ratio is most commonly used to readout FRET efficiency, which largely depends on the proximity between donor and acceptor. Here, we introduce an anisotropy-based mode of FRET detection (FADED: FRET-induced Angular Displacement Evaluation via Dim donor), which probes for relative orientation rather than proximity alteration. A key element in this technique is suppression of donor bleed-through, which allows measuring purer sensitized acceptor anisotropy. This is achieved by developing Geuda Sapphire, a low-quantum-yield FRET-competent fluorescent protein donor. As a proof of principle, Ca2+ sensors were designed using calmodulin as a sensing domain, showing sigmoidal dose response to Ca2+. By monitoring the anisotropy, a Ca2+ rise in living HeLa cells is observed upon histamine challenging. We conclude that FADED provides a method for quantifying the angular displacement via FRET., The FRET efficiency usually predominantly depends on the proximity of donor and acceptor. Here the authors report an anisotropy-based mode of FRET detection, FRET-induced Angular Displacement Evaluation via Dim donor (FADED), to allow quantification of the relative angle between donor and acceptor.

To compare CT findings of early (within 3 weeks post-onset)- and later (within 1 month before or after diagnostic criteria were satisfied, and later than 3 weeks post-onset) stage thrombocytopenia, anasarca, fever, reticulin fibrosis, renal dysfunction, and organomegaly (TAFRO) syndrome. Between 2014 and 2019, 13 patients with TAFRO syndrome (8 men and 5 women; mean age, 54.9 years) from nine hospitals were enrolled. The number of the following CT findings (CT factors) was recorded: the presence of anasarca, organomegaly, adrenal ischaemia, anterior mediastinal lesion, bony lesion, and lymphadenopathy. Records of adrenal disorders (adrenomegaly, ischaemia, and haemorrhage) throughout the disease course were also collected. Differences in CT factors at each stage were statistically compared between remission and deceased groups. Para-aortic oedema and mild lymphadenopathy were observed in all patients, whereas pleural effusion, ascites, and subcutaneous oedema were found in 5/13, 7/13, and 7/13 cases, respectively, at the early stage. CT factors at the early stage were significantly higher in the deceased than in the remission group (mean, 11 vs 6.5; p = 0.04), while they were nonsignificant at the later stage. Adrenal disorders were present in 7/13 cases throughout the course including 6 of adrenomegaly and 4 of ischaemia at the early stage. Para-aortic oedema and mild lymphadenopathy are most common at the early stage. Anasarca, organomegaly, lymphadenopathy, and adrenal disorders on early-stage CT are useful for unfavourable prognosis prediction. Moreover, adrenal disorders are frequent even at the early stage and are useful for early diagnosis of TAFRO syndrome. • CT findings facilitate early diagnosis and prognosis prediction in TAFRO syndrome. • Adrenal disorders are frequently observed in TAFRO syndrome. • Adrenal disorders are useful for differential diagnosis of TAFRO syndrome.

LSSmOrange is a fluorescent protein that exhibits a large energy gap between absorption and emission, which makes it a useful tool for multicolor bioimaging. This characteristic of LSSmOrange originates from excited-state proton transfer (ESPT): The neutral chromophore is predominantly present in the ground state while the bright fluorescence is emitted from the anionic excited state after ESPT. Interestingly, it was reported that this ESPT process follows bimodal dynamics, but its origin has not clearly been understood. We investigate ESPT of LSSmOrange using time-resolved impulsive stimulated Raman spectroscopy (TR-ISRS) that provides femtosecond time-resolved Raman spectra. The results indicate that the bimodal ESPT dynamics originates from the structural heterogeneity of the chromophore. Species-associated Raman spectra obtained by spectral analysis based on singular value decomposition (SVD) suggest that cis and trans chromophores coexist in the ground state. It is considered that these two forms are photoexcited and undergo ESPT in parallel, resulting in the bimodal dynamics of ESPT in LSSmOrange.

A new landslide cannot form until the soil has recovered to the critical depth for the recurrence of a landslide through the weathering of bedrock and soil transportation from adjacent areas. In volcanic areas with tephra deposits, landslides expose tephra and not bedrock. Therefore, the immunity of landslides in volcanic areas may be different from that of landslides in non-volcanic areas. Herein, we developed landslide inventory maps (LIMs) for 6 periods during 1955–2016 using aerial photographs and digital elevation models in northern Aso Volcano. In this area, landslides were found to continuously occur due to rainfall events and a large earthquake. Using the 6 LIMs, we examined the terrain attributes (i.e., slope angle, slope aspect, and normalized distance to ridge) and the overlap of landslides. Among the terrain attributes, slope angle was a dominant factor affecting the occurrence of landslides caused by both rainfall events and an earthquake. The total landslide areal density in 2016 was 50% for a slope angle of 35%–45%. 2 atypical events (a rainfall in July 2012 and an earthquake in April 2016) caused landslides to occur on slopes that were relatively resistant to landslides by typical amounts of rainfall, resulting in high landslide density in 2016. The intensity of rainfall for an event in July 2012 was considerably higher than that for other rainfall events. The type of landslides caused by an earthquake in April 2016 was different from that of landslides caused by rainfall. The depths of some landslides caused by this earthquake were deeper than those of landslides caused by the rainfall in July 2012. The overlap ratio was

Most targeting strategies of anticancer drug delivery systems (DDSs) rely on the surface functionalization of nanocarriers with specific ligands, which trigger the internalization in cancer cells via receptor-mediated endocytosis. The endocytosis implies the entrapment of DDSs in acidic vesicles (endosomes and lysosomes) and their eventual ejection by exocytosis. This process, intrinsic to eukaryotic cells, is one of the main drawbacks of DDSs because it reduces the drug bioavailability in the intracellular environment. The escape of DDSs from the acidic vesicles is, therefore, crucial to enhance the therapeutic performance at low drug dose. To this end, we developed a multifunctionalized DDS that combines high specificity towards cancer cells with endosomal escape capabilities. Doxorubicin-loaded mesoporous silica nanoparticles were functionalized with polyethylenimine, a polymer commonly used to induce endosomal rupture, and hyaluronic acid, which binds to CD44 receptors, overexpressed in cancer cells. We show irrefutable proof that the developed DDS can escape the endosomal pathway upon polymeric functionalization. Interestingly, the combination of the two polymers resulted in higher endosomal escape efficiency than the polyethylenimine coating alone. Hyaluronic acid additionally provides the system with cancer targeting capability and enzymatically controlled drug release. Thanks to this multifunctionality, the engineered DDS had cytotoxicity comparable to the pure drug whilst displaying high specificity towards cancer cells. The polymeric engineering here developed enhances the performance of DDS at low drug dose, holding great potential for anticancer therapeutic applications. ispartof: SCIENTIFIC REPORTS vol:9 issue:1 ispartof: location:England status: published

Mixed micelles based on amphiphilic gadolinium(III)-DOTA and europium(III)-DTPA complexes were synthesized and evaluated for their paramagnetic and optical properties as potential bimodal contrast agents. Amphiphilic folate molecule for targeting the folate receptor protein, which is commonly expressed on the surface of many human cancer cells, was used in the self-assembly process in order to create nanoaggregates with targeting properties. Both targeted and nontargeted nanoaggregates formed monodisperse micelles having distribution maxima of 10 nm. The micelles show characteristic europium(III) emission with quantum yields of 2% and 1.1% for the nontargeted and targeted micelles, respectively. Fluorescence microscopy using excitation at 405 nm and emission at 575-675 nm was employed to visualize the nanoaggregates in cultured HeLa cells. The uptake of folate-targeted and nontargeted micelles is already visible after 5 h of incubation and was characterized with the europium(III) emission, which is clearly observable in the cytoplasm of the cells. The very fast longitudinal relaxivity r

Background: Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC) refractory to gemcitabine + nab-paclitaxel (GN) due to suppression of cancer associated fibroblast (CAF) and increase of drug infiltration in tumor. We sought to determine the safety, recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with GN-refractory MPC. Methods: This phase 1, dose finding trial was conducted following preclinical study of IL-6 signaling blockade in genetically engineered mouse model of pancreatic cancer (pGEMM) and patients-derived CAF, enrolled 10 patients with MPC that had progressed after GN. During the dose-finding part to determine dose-limiting toxicity (DLT), patients initially received TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. The subsequent dose-expansion part used the TCZ dose identified in the dose-finding study. Before Cycle 1 and at the end of Cycle 1, biopsy of liver metastasis was performed 3 to 5 hours after levofloxacin (LVXF) administration to measure LVFX infiltration in tumor using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). Results: In pGEMM tumor, mouse IL-6 receptor antibody plus GN led pathological response. The growth of CAFs from patients with pancreatic cancer was inhibited by TCZ. In this phase I study, no DLTs occurred and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events (TEAEs) occurred in 80% of patients (Grade ≥3 in 60%) with decreased neutrophil count (n=5, 7 events) being the most common TEAE. Disease control rate was 80·0% (95%CI: 44·4-97·5) and tumor reduction during cycle 1 was observed in 4 patients who were defined as responder. In paired-biopsied samples, decrease of phosphorylated (p) STAT3 expression in tumor was observed in 7 of 8 patients (88%). Responder-related biological activities were increase of cleaved PARP expression of tumor nuclei (P = 0.01), decrease of proliferative CAF (P = 0.08), and increase of LVFX infiltration in tumor (P = 0.04). Decrease of pSTAT3 expression (P = 0.02) was favor to increase of LVFX infiltration against increase of gamma-H2AX, an index of gemcitabine exposure (P = 0.20). Conclusion: TCZ+GN-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and gain of intratumoral drug infiltration in MPC. Citation Format: Shuichi Mitsunaga, Masafumi Ikeda, Kazunori Aoki, Kyoko Yamaguchi, Noriaki Sawada, Etsuko Fujii, Masanobu Nishidate, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Atsuhiko Kato, Mayu Makikawa, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT565.

Background: Blockade of interlukine-6 (IL-6) and programmed death-1-ligand 1 (PD-L1) limits tumour progression in murine models of pancreatic cancer. When PD-L1 expression of metastatic pancreatic cancer (MPC) is increased under the influence of gemcitabine exposure plus IL-6 inhibition, the spectrum of benefit from immune checkpoint blockade may expand to MPC patients with combined blockade of IL-6 and PD-L1 plus gemcitabine-based chemotherapy. We sought to determine PD-L1 expression of liver metastasis during cycle 1 of a phase I study, tocilizumab (TCZ) plus gemcitabine/nab-paclitaxel (GN) in patient with GN-refractory MPC. Methods: This phase 1 study enrolled 10 patients with MPC that had progressed after GN. All patients experienced cycle 1 of TCZ plus GN, TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. Biopsy of liver metastasis was performed before Cycle 1 and at the end of Cycle 1. Tumour microenvironment including PD-L1 expression were evaluated in paired-biopsied specimen from 7 patients by multicolour immunofluorescence staining. Tumour cell and PD-L1 expression were labelled by anti-cytokeratin 19 antibody and anti-PD-L1 antibody clone SP263, respectively. Cell numbers per millimetre squared (cell density) and the percentage of PD-L1 positive cell in tumor area were calculated according to cell types including tumour cell as cytokeratin 19 positive cell and macrophage as CD68 positive cell. Results: Cell density and the percentage of PD-L1 positive tumor cell were 29/mm2 and 1.1% in mean at baseline and tended to be increased up to 142/mm2 (P = 0.11) and 5.2% (P = 0.06) at the end of cycle 1. Means of PD-L1 positive macrophages were 28/mm2 and 6.9% at baseline and were increased to 69/mm2 (P = 0.01) and 14% (P = 0.08). When region of interest was selected from tumor stroma, differences of PD-L1 positive macrophage during cycle 1 was also maintained in cell density (P = 0.02) and the percentage of PD-L1 positive cell (P = 0.06). Conclusion: TCZ+GN induced an increase of PD-L1 expression levels on tumor cell and macrophages. Citation Format: Kyoko Yamaguchi, Etsuko Fujii, Shuichi Mitsunaga, Noriaki Sawada, Masafumi Ikeda, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Mayu Makikawa, Kazunori Aoki, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Atsukiko Kato, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Increased PD-L1 expression levels were observed on both tumor cells and macrophages by tocilizumab plus gemcitabine/nab-paclitaxel treatment in gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6145.

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson’s and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via single-channel on-cell patch clamp we show heteropentameric conductances resemble those of the α3K splice variant. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR α3 channels, which is important to understand and tackle α3 related diseases.SummaryThe glycine receptor α3 is key to the central nervous system’s physiology and involved in chronic pain and epilepsy. In this paper, Lemmens et al. reveal and functionally characterize α3 splice variant heteropentamerization via advanced single-molecule fluorescence image analysis.DeclarationsFundingWe acknowledge the UHasselt Advanced Optical Microscopy Centre (AOMC). Prof. Em. Marcel Ameloot, the Research Foundation Flanders (FWO, project G0H3716N) and the province of Limburg (Belgium) (tUL Impuls II) are acknowledged for funding the microscopy hardware. V. Lemmens is grateful for a doctoral scholarship from the UHasselt (17DOC11BOF) and KU Leuven (C14/16/053) Special Research Funds (BOF).Conflicts of interest / competing interestsNo conflicts of interest apply.Ethics approvalNot applicableAvailability of data and materialAll data and material are available upon request.Code availabilityFluctuation imaging and co-localization analyses were performed in the software package PAM [71]. The software is available as source code, requiring MATLAB to run, or as pre-compiled standalone distributions for Windows or MacOS athttp://www.cup.uni-muenchen.de/pc/lamb/software/pam.htmlor hosted in Git repositories underhttp://www.gitlab.com/PAM-PIE/PAMandhttp://www.gitlab.com/PAM-PIE/PAMcompiled. A detailed user manual is available athttp://pam.readthedocs.io.Author contributionsConceptualization Meier J.C., Brône B. and Hendrix J.; Investigation and formal analysis Lemmens V. and Thevelein B.; Software development Hendrix J.; Writing the original draft Lemmens V., Thevelein B and Hendrix, J.; Review and editing by all authors.

Somatic mutation of DNMT3A (DNA methyltransferase 3 alpha) is implicated in the development of a wide range of hematological disorders, including clonal hematopoiesis of indeterminate potential. To elucidate the functional roles of endogenous levels of a DNMT3A R882 mutant, we generated a novel Dnmt3a R878C conditional knock-in mouse model. In contrast to viable heterozygotes, mice homozygous for the Dnmt3a R878C mutation in the hematopoietic system were not viable (Dnmt3a R878C is homologous to human DNMT3A R882C). Hematopoietic cell-specific heterozygous expression of Dnmt3a R878C led to significant expansion of adult quiescent hematopoietic stem cells (HSCs); however, these mice had no hematological malignancies. The expanding HSC population in heterozygous Dnmt3a R878C knock-in mice had an accumulation of G0-phase cells. In contrast to aberrantly enhanced self-renewal capacity in vitro, heterozygous Dnmt3a R878C knock-in HSCs had no competitive repopulating advantage in vivo over wild-type HSCs. Considering the capacity of the heterozygous Dnmt3a R878C mutant for HSC pool expansion, our Dnmt3a R878C knock-in mouse line is a useful platform on which to dissect the pathophysiology of clonal hematopoiesis. This mouse line can also help to elucidate the biological and molecular actions of DNMT3A mutations in the malignant transformation of normal HSCs.

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane. ispartof: ELIFE vol:9 ispartof: location:England status: published

This study aimed to elucidate the effects of a liquid polymeric formula containing alginate, a source of water- soluble dietary fiber, on stool form, intestinal microbiota, and the production of short-chain fatty acid production. Participants were 11 elderly patients with malnutrition and poor oral intake, who required enteral feeding. The alginate-containing liquid formula, which characteristically changes state from liquid to semi-solidified depending on pH, was administered for 4 weeks. This variable-viscosity liquid formula significantly improved the values of nutritional indices and stool form. Analysis of fecal specimens revealed significant decreases in pH. The concentrations of short-chain fatty acids did not change significantly in stool samples, but acetic acid and propionic acid were significantly increased in blood samples. The proportion of Clostridium cluster XI in fecal microbiota was also significantly increased. The alginate-containing variable-viscosity liquid formula significantly improved stool form, lowered fecal pH, and short-chain fatty acid concentrations in blood.

Background Myelofibrosis (MF) is a myeloproliferative neoplasm which is associated with megakaryocytic atypia, fibrosis in the bone marrow (BM), and extramedullary hematopoiesis, followed by progressive hematopoietic failure and leukemic transformation. JAK1/JAK2 inhibitors are currently available and reduce spleen volume, improve symptoms related to MF and prolong the overall survival (OS). Although the benefits associated with JAK1/JAK2 inhibitors are well established, not all patients respond to these inhibitors, and long-term exposure to these inhibitors results in the emergence of resistant clones based on the reactivation of the JAK-STAT pathway. Therefore, new therapeutic strategies targeting other molecules can provide additional benefits to patients with MF. In the previous study, we identified Calcium/Calmodulin Dependent Protein Kinase II Gamma (CAMK2G) as a new therapeutic target for MF by performing compound screening. Furthermore, CAMK2G inhibition can overcome drug-resistance against JAK2 inhibitors. Therefore, it is important to investigate the mechanisms underlying the therapeutic effect of CAMK2G inhibition. In this study, to explore the mechanism underlying the therapeutic effect of CAMK2G inhibition, we performed the immunoprecipitation mass spectrometry to find out the protein that has the direct interaction with CAMK2G. Methods Quantitative Proteomic Analysis of the Target Proteins of CAMK2G The protein complexes with FLAG-tagged CAMK2G (FLAG-CAMK2G) were trapped by anti-FLAG antibody. The eluted assay mixtures were reduced, alkylated and digested into peptides. Each peptide solution and control mixture were labeled with differential stable isotope tags. The samples were quantitatively analyzed using a Q Exactive mass spectrometer (Thermo Fisher Scientific). The spectra were searched against the SWISS-PROT databases using SEQUEST on Proteome Discoverer software 2.2 (Thermo Scientific). Results To reveal the , we conducted the immunoprecipitation assays by FLAG-CAMK2G or FLAG-tag alone, overexpressed in MF model cells. Because CAMK2G has kinase activity, we attempted to reveal the kinase-activity-dependent targets using unhydrolyzable ATP analog, AMP-PNP（5'-adenylyl-imidodiphosphate）that can maintain the strong-binding state of kinase with its target. The protein complexes with FLAG-CAMK2G were trapped by anti-FLAG antibody. After samples were digested into peptides and the candidate proteins were identified and quantitatively analyzed by mass spectrometry. As a result, we identified eukaryotic translation initiation factor 4B (eIF4B) as a protein that has a direct interaction with CAMK2G. eIF4B is a part of the complex involved in the initiation of translation. It has been reported that eIF4B plays a role in the translation of factors involved in anti-apoptosis (Bcl2, Bclxl, Mcl1), cell cycle (Cdc25c), and cell proliferation (c-Myc). We then checked whether knockdown of eIF4B decrease proliferation of MF model cell line. shRNA-mediated silencing of eIF4B decreased cell growth of these cells. Furthermore, the phosphorylation of eIF4B was increased by the ectopic expression of MPL W515L, one of the common mutations found in MF. Also, the phosphorylation of eIF4B was increased by the overexpression of CAMK2G. We then explored the proteins regulated by eIF4B in MF and identified that knockdown of eIF4B decreased the amount of Bcl2 protein. Conclusion In our study, we performed immunoprecipitation mass spectrometry and identified eIF4B as a partner protein of CAMK2G. Since CAMK2G inhibition was shown to be effective against MF in vitro and in vivo, we focused on eIF4B as a potential effector in MF. We also showed that overexpression of MPL W515L and CAMK2G phosphorylates eIF4B and that knockdown of eIF4B inhibited proliferation of MF cells. Furthermore, Bcl2 can be one of the target proteins regulated by eIF4B. Based on these, eIF4B plays an important role in MF. We further perform ribosome profiling to comprehensively understand the regulation of translation by eIF4B. Our research not only elucidate the pathogenesis of MF but also identify new therapeutic targets for MF. Disclosures Masamoto: MSD K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Kurokawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau.

Background: Erdheim-Chester disease (ECD) is a type of systemic, non-Langerhans histiocytic disorder characterized by diffuse organ damage with infiltration of CD68-positive, CD163-positive, and CD1a-negative histiocytes. Although most patients have mutations associated with the hyperactivation of the MAPK pathway, including BRAF, MAP2K1, and NRAS, the remaining have no known driver mutations. So far, there is no specific target of treatment for them. To elucidate novel driver mutations and establish new treatment strategies in these patients, we performed whole-exome sequencing (WES) and whole-genome sequencing (WGS) using patient samples with ECD. In addition, we performed WES with multi-organ lesions in a patient with ECD to reveal the mutational profiles of each organ lesion. Methods: We performed a nationwide survey and collected clinical samples of ECD in Japan. We collected 22 samples of ECD lesions from 15 adult patients. All cases were pathologically proved. Twenty of 22 samples were formalin-fixed and paraffin-embedded tissue (FFPE) and were examined by WES. For WGS, DNA was extracted from 2 raw samples of ECD lesions. In addition, we collected multi-organ lesions in 3 patients. One patient developed myelodysplastic syndrome (MDS) and subsequent acute myeloid leukemia (AML). Therefore, we also performed WES with each bone marrow (BM) sample to reveal the relationship between ECD and these myeloid malignancies. We used 6 samples of peripheral blood, 1 BM sample, 1 skin sample, and 1 oral mucosa sample as normal controls. Result: We detected known driver mutations in seven of 15 cases (47%). Among them, BRAF V600E was detected in 5 cases (8 samples), MAP2K1 C121S in 1 case (1 sample), and NRAS Q61R in 1 case (2 samples) by WES and WGS. A mean of 69 nonsynonymous mutations per patient was identified in normal-tumor analysis (range, 2-491) and 188 in tumor-only analysis (range, 17-3598) of WES, and 3134 in normal-tumor analysis (range, 2588-3680) of WGS. The median variant allele frequency (VAF) for the 11 samples with known activating kinase mutations identified by WES and WGS was 14.4% (range, 6.3-34.7). We could not detect known driver mutations in the other 8 cases (53%). Therefore, to reveal novel driver mutations, we focused on these 8 cases. Notably, EPHA2 P786L, MYBPC3 D798N, TDRD5 P115L, and TCEAL4 F17L mutations are recurrently found in 2 out of the 8 cases, suggesting new driver mutations are contained in these. The VAFs of EPHA2 P786L were 9.1% and 9.3%, MYBPC3 D798N 5.5% and 11.9%, TDRD5 P115L 10.2% and 18.7%, and TCEAL4 F17L 7.5% and 9.0% in each case. We also analyzed multiple organ samples of a case with BRAF V600E mutation (5 samples of ECD lesions, BM with MDS, BM with AML, and skin as normal control). Interestingly, BRAF V600E mutation was identified in 3 samples (bone lesion, heart, and intestine) but was not in other samples (kidney lesion, dura matter, BM tumor, BM with MDS, and BM with AML), suggesting mutational profiles are different depending on the organ of the lesion. Conclusion: Our nation-wide analysis revealed that no well-known driver mutations were found in more than half of the ECD cases. We identified EPHA2 P786L, MYBPC3 D798N, TDRD5 P115L, and TCEAL4 F17L mutations as candidates of novel driver mutations in ECD. To elucidate the role of these mutations in pathogenesis of ECD, further functional analyses are warranted. In addition, we revealed heterogeneity of mutational profile of multi lesions in an ECD patient with BRAF V600E mutation. It is noteworthy that mutational profiles might be different depending on the organ of the lesion. Disclosures Honda: Chugai Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee; Takeda Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee. Matsuda: Kyowa Kirin: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee. Taoka: ONO PHARMACEUTICAL CO., LTD.: Other; Chugai Pharmaceutical Company: Other. Kurokawa: Teijin Limited: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau.

Resistance to anthracycline and cytarabine based conventional chemotherapy often occurs and results in extremely poor prognosis in patients with acute myeloid leukemia (AML). Although chemotherapy resistance is the most critical clinical problem, the mechanisms by which AML confers resistance to conventional chemotherapy are not yet fully understood. In this study, we investigated the key mechanisms of chemotherapy resistance through single cell RNA-sequencing analysis using paired bone marrow AML cells longitudinally collected from two AML-MRC patients at diagnosis and relapse after anthracycline-based chemotherapy. AML blasts were sorted by CD45/SSC gating and subjected to single cell RNA-seq analysis. Single cell RNA-seq was performed using 10x Genomics' Chromium System. Mean estimated number of cells per sample was 3.403 (2,731-4,200) and median detected genes per cell ranged 3,030 to 3,918 among four samples. Data collected from paired samples were combined in following analysis. Transcriptome based clustering following UMAP dimensionality reduction distinguished 5 and 9 cluster groups in each paired sample. Chemotherapy sensitive cluster groups dominant at diagnosis and chemotherapy resistant cluster groups dominant at relapse were clearly divided. In each paired sample, a few AML cells at diagnosis were allocated to chemotherapy resistant cluster groups. This suggested that transcriptionally identifiable less frequent cells resistant to chemotherapy existed at diagnosis and may expand during and/or after chemotherapy maintaining its transcriptional features. Next, to determine whether these transcriptional features are correlated with DNA mutation profiles, we labeled DNA mutation status to each cell and compared frequencies of mutation. As far as we detected, AML recurrent mutations such as DNMT3A R882C and TP53 missense mutation were not related to chemotherapy resistant cluster groups, although this method was relatively limited by the nature of RNA-seq-based mutation detection. Then we sought to determine transcriptional features of resistant clones. Gene set enrichment analysis identified some gene groups such as E2F signaling pathway, MYC signaling pathway, hedgehog signaling pathway and TNFA signaling pathway as transcriptional signatures related to emergence after chemotherapy. Analysis of known hematopoietic differentiation gene signatures showed distinct differentiation profiles in each cluster groups, whereas resistant cluster groups were not necessarily related to hematopoietic stem cell signatures. Intrapatient variations of transcriptional signatures among the resistant cluster groups were detected, which indicated that accurate detection of transcriptional features related to chemotherapy resistance may be difficult by using bulk RNA-seq method. As for other cluster groups which were not dominant both at diagnosis and relapse, these cluster groups hardly changed its frequencies between at diagnosis and relapse, which suggested less proliferative leukemia cells persisted during chemotherapy and have various transcriptional features although whether these persisting cells contribute to relapse was unclear. Since enriched transcriptional signatures in resistant cluster groups were not consistent between the two patients, further analysis using samples collected from more patients would be needed to determine common critical chemotherapy resistant transcriptional signature. In conclusion, our analysis suggested that a transcriptionally identifiable small fraction of cells showing gene signatures related to chemotherapy resistance at diagnosis may expand during chemotherapy and revealed intrapatient transcriptional complexity of response to chemotherapy, which cannot be uncovered by bulk RNA-sequencing. Disclosures Honda: Takeda Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee. Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau.

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1 is indispensable for normal hematopoiesis. We have previously reported that EVI1 expression is restricted to hematopoietic stem cell fraction and EVI1-expressing cells show robust long-term reconstitution capacity using Evi1-IRES-GFP knock-in (EVI1-GFP) mice, which enable us to track Evi1 expression on a single cell basis. In this study, we tried to elucidate the functional implication of EVI1 expression in AML using these mice. We generated murine EVI1-GFP AML model by retrovirally transducing MLL-AF9 or -ENL fusion gene into Lineage- Sca-1+ c-kit+ (LSK) cells from EVI1-GFP mice followed by transplantation into lethally irradiated syngeneic mice. Clonogenic and leukemogenic potentials of AML cells, especially leukemic cells with a granulocyte-macrophage progenitor phenotype (L-GMPs) from these mice, were compared according to GFP expression. Remarkably, GFP-positive L-GMPs tended to show lower colony-forming activity in semi-solid media and lower leukemia-initiating potential than GFP-negative L-GMPs. GFP-positive L-GMPs, however, induced a more aggressive form of AML, characterized by shorter survival in the secondary transplantation model. When EVI1-GFP AML mice underwent cytotoxic chemotherapy with cytarabine, the GFP-positive fraction was enriched during myelosuppression, indicating the survival advantage of EVI1-positive cells. To investigate the downstream target of EVI1, we employed murine EVI1-AML model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we performed chromatin-immunoprecipitation coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody. To identify leukemia-specific targets of EVI1, the result was compared with the result of ChIP-seq obtained from 32D-cl3 murine hematopoietic progenitor cells with 3×FLAG tag inserted into 3'-end of the coding region of the EVI1 gene. Gene ontology analysis revealed that genes involved in immune processes are explicitly enriched in the leukemia samples. Among the list of EVI1-bound genes, we tried to refine functional downstream targets of EVI1, which are upregulated in murine EVI1-AML cells and of which expressions are positively correlated with EVI1. By combining the ChIP-seq data with murine transcriptome data that compare hematopoietic progenitor cells expressing empty-vector and EVI1+ AML cells, and public gene expression datasets of human AML (Valk et al. NEJM 2004), we picked out 18 genes as candidate EVI1 downstream genes. Functional screening using EVI1-AML cells and shRNAs against these genes revealed that silencing of ETS transcription factor ERG (ETS-related gene) markedly suppressed proliferation and colony-forming activity of EVI1-AML cells, as well as rendered them vulnerable to cytotoxic agents. Normal c-kit+ hematopoietic progenitor cells were less affected by shRNAs against ERG. By comparing MLL-ENL immortalized murine hematopoietic cells with high and low EVI1 expression, EVI1-high MLL-ENL cells showed higher ERG dependency than EVI1-low MLL-ENL cells. Pharmacological inhibition of ERG also led to marked inhibition of EVI1-AML cells and EVI1-high MLL-ENL cells. Finally, knockdown of ERG remarkably delayed AML development in bone marrow transplantation model of EVI1-AML and EVI1-expressing MLL-ENL AML. Our data suggest that EVI1-positive AML cells are characterized by an aggressive nature and resistance to cytotoxic agents, as well as low leukemia stem cell capacity. ERG would be a novel downstream target of EVI1, on which survival of EVI1-expressing AML cells depends. Disclosures Masamoto: Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; MSD K.K.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau. Kurokawa: Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau.

Background: Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), was previously identified as expressed by mRNA in patients with multiple myeloma with only low expression detected in normal tissues, but confirmation of protein expression remained elusive. In this study, we determined the cell surface expression of GPRC5D on malignant and normal hematological cells. In addition, we evaluated the antitumor activity and mechanism of GPRC5D TRABs in in vitro and in vivo mouse models. Method & Results: We established specific monoclonal antibodies against human GPRC5D and identified its expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, did not find it at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. To investigate whether GPRC5D could be therapeutic target, we generated IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRABs). GPRC5D TRABs induced T cell activation and the killing of a wide variety of GPRC5D expressing tumor cells in vitro. In mouse models with reconstituted human immune cells, GPRC5D TRABs showed strong antitumor efficacy against GPRC5D-positive tumors through the activation of T cells. Conclusion: These findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy. Citation Format: Tatsushi Kodama, Yu Kochi, Waka Nakai, Hideaki Mizuno, Takeshi Baba, Kiyoshi Habu, Noriaki Sawada, Hiroyuki Tsunoda, Takahiro Shima, Kohta Miyawaki, Yoshikane Kikushige, Yasuo Mori, Toshihiro Miyamoto, Takahiro Maeda, Koichi Akashi. Anti -GPRC5D/CD3 T cell-redirecting bispecific antibody with potent in vitro and in vivo antitumor efficacy against multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1886.

Investigation of protein-protein interactions (PPI) in Candida albicans is essential for understanding the regulation of the signal transduction network that triggers its pathogenic lifestyle. Unique features of C. albicans, such as the alternative codon usage and incomplete meiosis, have enforced the optimization of standard genetic methods as well as development of novel approaches. Since the existing methods for detection of PPI are limited for direct visualization of the interacting complex in vivo, we have established a bimolecular fluorescence complementation (BiFC) in C. albicans, a powerful technique for studying PPI. We have developed an optimized set of plasmids that allows for N- and C-terminal tagging of proteins with split yeast-enhanced monomeric Venus fragments, so that all eight combinations of fusion orientations can be analyzed. With the use of our BiFC assay we demonstrate three interaction complexes in vivo, which were also confirmed by two-hybrid analysis. Our Candida optimized BiFC assay represents a useful molecular tool for PPI studies and shows great promise in expanding the knowledge on molecular mechanisms of protein functions. ispartof: G3-Genes Genomes Genetics vol:7 issue:10 pages:3509-3520 ispartof: location:England status: published