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Keratinized lesions have been a conceivable false-negative (FN) factor in oral exfoliative cytology (OEC); however, other factors are poorly analyzed. In this study, we aimed to identify the factors influencing the accuracy of OEC and FNs focusing on the lesion characteristics, patient background, and surgeon factors in oral potentially malignant disorders (OPMD).We retrospectively studied 44 patients who underwent both OEC and histopathological diagnosis. Sensitivity, specificity, FN rate, false-positive (FP) rate, and prevalence of both methods were compared. Similarly, accuracy indices were compared among clinical diagnosis groups (leukoplakia vs. other diagnosis). The association between patient and surgeon-related factors influencing FN OEC results were investigated using Fisher's exact test and a multiple logistic regression analysis.Overall, the sensitivity; specificity; and FN, FP, and prevalence rates of OEC were 31.8%, 82.1%, and 68.8%, 17.9%, and 36.4%, respectively. Leukoplakia was significantly more common in clinical diagnosis (P = 0.007) with sensitivity, specificity, and FN rates of 20.0%, 95.2%, and 80.0%, respectively. Contrarily, non-keratinized lesions had sensitivity, specificity, and FN of 83.3%, 85.7%, and 16.7%, respectively. In the prevalent group, leukoplakia and anucleate squamous cells were significantly associated with FN cases (P = 0.013, P = 0.050). On multivariate analysis in OEC negative patients, age ≤64 (P = 0.050) and location on the tongue (P = 0.047) was independently associated with FNs.FN of OEC was conceivable to be due to poor deep-seated cell sampling, which was associated with leukoplakia, age, and location. Therefore, these factors may be considered in the evaluation of OEC results.

Context J-DISCOVER is a prospective observational cohort study aiming to understand the current management of patients with early-stage type 2 diabetes mellitus (T2DM) in Japan, enrolling patients initiating second-line treatment. Objective The current analysis examined the change in treatment satisfaction during the study period and factors affecting this change among patients in J-DISCOVER. Methods We used data from the J-DISCOVER study, in which 1798 patients with T2DM aged ≥ 20 years were enrolled from 142 sites across Japan. Treatment satisfaction was assessed using the Diabetes Treatment Satisfaction Questionnaire (DTSQ). Results The mean DTSQ treatment satisfaction score increased from 25.9 points at baseline to 27.3 points at 6 months, which was maintained through 36 months. Among the baseline characteristics examined, higher baseline DTSQ treatment satisfaction scores (P Conclusion Changes in DTSQ treatment satisfaction scores were related to HbA1c improvement, suggesting that the management strategy was appropriately planned for each patient. The results also suggest that the availability of social support for patients with T2DM who are elderly or living alone may be an important factor affecting treatment satisfaction, adherence, and clinical outcomes.

Many patients with type 2 diabetes mellitus (T2DM) suffer from complications that impose substantial burdens on prognosis and medical costs. Accumulating evidence has demonstrated the clinical benefit of sodium-glucose cotransporter 2 inhibitors (SGLT2i) on cardiovascular and renal complications. However, the health economic impact of SGLT2i remains unclear. The aim of this study was to evaluate the cost-effectiveness of initiating antidiabetic therapy with an SGLT2i using Japanese real-world data.We constructed a natural history model incorporating heart failure (HF), myocardial infarction, stroke, chronic kidney disease, and end-stage renal disease (ESRD) as complications. The target population comprised patients with T2DM who newly initiated their first oral glucose-lowering drugs. By using a population-based microsimulation, we estimated the 10-year medical costs in Japanese yen (JPY) and outcomes (hospitalization for/development of complications and quality-adjusted life years [QALY]) for patients who initiated antidiabetic therapy with an SGLT2i or conventional therapy. Sensitivity analyses included a probabilistic sensitivity analysis (PSA) with 1,000,000 iterations.In the base-case analysis, the total medical cost per person was JPY 1,638,806 versus JPY 1,825,033 and the QALYs were 8.732 versus 8.513 for the SGLT2i strategy versus the conventional strategy, respectively. Thus, initiating treatment with an SGLT2i was dominant, more effective (QALY gain), and lower cost. When treating 10,000 patients, the SGLT2i strategy would reduce all-cause deaths by 410 (552 vs 962), HF events by 201 (897 vs 1098), and ESRD events by 16 (16 vs 32) versus the conventional strategy. The PSA revealed that the probability of dominance for initiating SGLT2i therapy was 90.5%, demonstrating the robustness of the results.Our results suggest that initiating T2DM treatment with SGLT2i, aimed at managing cardiovascular and renal complications from the early stages of diabetes, can improve the clinical outcome and reduce cost burden of T2DM.

Resistance to apoptosis in acute myeloid leukemia (AML) cells causes refractory or relapsed disease, associated with dismal clinical outcomes. Ferroptosis, a mode of non-apoptotic cell death triggered by iron-dependent lipid peroxidation, has been investigated as potential therapeutic modality against therapy-resistant cancers, but our knowledge of its role in AML is limited. We investigated ferroptosis in AML cells and identified its mitochondrial regulation as a therapeutic vulnerability. GPX4 inhibition induces ferroptosis in AML cells, accompanied with characteristic mitochondrial lipid peroxidation, exerting anti-AML effects in vitro and in vivo. Electron transport chains (ETC) are primary sources of coenzyme Q10 (CoQ) recycling for its function of anti–lipid peroxidation in mitochondria. We found that the mitochondria-specific CoQ potently inhibited GPX4 inhibition–mediated ferroptosis, suggesting that mitochondrial lipid redox regulates ferroptosis in AML cells. Consistently, Rho0 cells, which lack functional ETC, were more sensitive to GPX4 inhibition–mediated mitochondrial lipid peroxidation and ferroptosis than control cells. Furthermore, degradation of ETC through hyperactivation of a mitochondrial protease, caseinolytic protease P (ClpP), synergistically enhanced the anti-AML effects of GPX4 inhibition. Collectively, our findings indicate that in AML cells, GPX4 inhibition induces ferroptosis, which is regulated by mitochondrial lipid redox and ETC.

To assess the initial manifestation of comorbidities and their impact on mortality risk in patients with type 2 diabetes mellitus (T2DM) without a history of cardiovascular or renal complications (i.e., in the early stages of T2DM) compared with patients without T2DM.We performed a retrospective cohort study using a Japanese hospital claims database. The incidence rates of comorbidities (chronic kidney disease [CKD], heart failure [HF], myocardial infarction [MI], peripheral arterial disease [PAD], and stroke) and mortality risk were compared between patients with T2DM and age-/sex-matched patients without T2DM (matched 1:2).Among the comorbidities assessed in this study, CKD and/or HF was the most frequent initial manifestation in the patients with T2DM (n = 426,186) with an incidence rate 2.02 times greater than that in matched patients without T2DM (n = 1,018,609). The mortality risk was also greater in patients with T2DM than in patients without T2DM with a hazard ratio of 1.73. In both patients with and without T2DM, the presence of CKD or HF was associated with greater mortality risks compared with the presence of MI, PAD, or stroke.The high incidence of CKD or HF manifestation can contribute to the augmented mortality risk in patients in the early stages of T2DM compared with patients without T2DM. These findings highlight the importance of early interventions for preventing/treating CKD and HF to improve the prognosis of patients with T2DM.

Cell migration is essential for many physiological and pathological processes, including embryonic development, wound healing, immune response and cancer metastasis. Inka2 transcripts are observed in migrating cells during embryonic development, suggesting the involvement of inka2 in cell migration. However, its precise role remains unclear. Here, we found that inka2 controlled focal adhesion dynamics and cell migration, likely by regulating protein phosphatase-2A (PP2A) function. A scratch assay revealed that inka2 shRNA-transfected NIH3T3 cells showed rapid wound closure, indicating an inhibitory effect by inka2 on cell migration. Live-cell imaging of NIH3T3 cells expressing EGFP-paxillin using total internal reflection fluorescence microscopy revealed that inka2 knockdown increased the turnover rate of focal adhesions. Given that PP2A, which consists of catalytic (C), regulatory (B) and scaffolding (A) subunits, is known to regulate focal adhesions, we examined the inka2-PP2A interaction. Immunoprecipitation revealed an association between inka2 and the PP2A C subunit. Binding of Inka2 to the C subunit prevented the association between the A and C subunits, suggesting that inka2 can inhibit PP2A function. Furthermore, both inka2 expression and PP2A inhibition decreased focal adhesion kinase-paxillin interaction, resulting in reduced formation of focal adhesions. We assessed the effect of pharmacological PP2A inhibition on the inka2 knockdown-induced increase in cell migration speed and found that treatment with a PP2A inhibitor negated the accelerated migration of inka2 knockdown cells. These results suggest that inka2 knockdown exerts its effects through PP2A-dependent regulation of focal adhesions. Our findings contribute to a better understanding of the molecular mechanisms underlying cell migration.

The lipoprotein particle number (PN) profile may be a better marker of cardiovascular risks than standard serum lipid measurements. The aim of this study was to analyze the lipoprotein PNs in Japanese children with abdominal obesity and to determine the subclass profile.The participants included 164 Japanese children (79 boys and 85 girls) aged 9-13 years. We obtained waist-to-height ratios (WHtR) and serum lipids for all participants. The lipoprotein PNs in 12 subclasses were analyzed using high performance liquid chromatography (HPLC).Both boys and girls with abdominal obesity (WHtR ≧ 0.5) had significantly higher triglyceride (TG), very-low-density lipoprotein (VLDL)-PN, and all VLDL-subclass PNs compared to those without abdominal obesity. In boys with abdominal obesity, low-density lipoprotein (LDL)-PN was higher, but lipoprotein cholesterol (LDL-C) was not, and high-density lipoprotein cholesterol (HDL-C) was lower, but HDL-PN was not compared to those without abdominal obesity. In girls with abdominal obesity, LDL-C and LDL-PN were not significantly different and both HDL-C and HDL-PN were lower compared to those without abdominal obesity. Subclass analyses demonstrated that boys and girls with abdominal obesity had significantly lower very large and large HDL-PNs than those without abdominal obesity. In addition, medium, small, and very small LDL-PNs were higher in boys with abdominal obesity than those without abdominal obesity.This study found that Japanese children with abdominal obesity are affected by the lipoprotein-subclass PN profile, with sex differences in the LDL- and HDL-subclasses, which is different from results obtained by standard serum lipid measurements.

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former conferring a poor prognosis. We have recently revealed that FLT3-ITD confers resistance to the PI3K/AKT pathway inhibitors by protecting the mTORC1/4EBP1/Mcl-1 pathway through Pim kinases induced by STAT5 activation in AML. The proteasome inhibitor bortezomib has recently been reported as a promising agent for treatment of AML. Here, we show that the proteasome inhibitor bortezomib as well as carfilzomib induces apoptosis through the intrinsic pathway more conspicuously in cells transformed by FLT3-TKD than FLT3-ITD. Mechanistically, bortezomib upregulated the stress-regulated protein REDD1 and induced downregulation of the mTORC1 pathway more distinctively in cells transformed by FLT3-TKD than FLT-ITD, while overexpression of Pim-1 partly prevented this downregulation and apoptosis in FLT3-TKD–transformed cells. Genetic enhancement of the REDD1 induction or pharmacological inhibition of STAT5, Pim kinases, mTORC1, or S6K by specific inhibitors, such as pimozide, AZD1208, PIM447, rapamycin, and PF-4708671, accelerated the downregulation of mTORC1/Mcl-1 pathway to enhance bortezomib-induced apoptosis in FLT3-ITD–expressing cells, including primary AML cells, while overexpression of Mcl-1 prevented induction of apoptosis. Thus, FLT3-ITD confers a resistance to the proteasome inhibitors on AML cells by protecting the mTORC1/Mcl-1 pathway through the STAT5/Pim axis, and inhibition of these signaling events remarkably enhances the therapeutic efficacy.

Ruxolitinib (RUX), a JAK1/2-inhibitor, is effective for myeloproliferative neoplasm (MPN) with both JAK2V617 F and calreticulin (CALR) mutations. However, many MPN patients develop resistance to RUX. Although mechanisms of RUX-resistance in cells with JAK2V617 F have already been characterized, those in cells with CALR mutations remain to be elucidated. In this study, we established RUX-resistant human cell lines with CALR mutations and characterized mechanisms of RUX-resistance. Here, we found that RUX-resistant cells had high levels of MPL transcripts, overexpression of both MPL and JAK2, and increased phosphorylation of JAK2 and STAT5. We also found that mature MPL proteins were more stable in RUX-resistant cells. Knockdown of MPL in RUX-resistant cells by shRNAs decreased JAK/STAT signaling. Immunoprecipitation assays showed that binding of mutant CALR to MPL was increased in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells. When resistant cells were cultured in the absence of RUX, the RUX-resistance was reversed, with reduction of the mutant-CALR/MPL complex. In conclusion, MPL overexpression induces higher levels of a mutant-CALR/MPL complex, which may cause RUX-resistance in cells with CALR mutations. This mechanism may be a new therapeutic target to overcome RUX-resistance.

Summary During neurodevelopment, the growth cone deciphers directional information from extracellular guidance cues presented as shallow concentration gradients via signal amplification. However, it remains unclear how the growth cone controls this amplification process during its navigation through an environment in which basal cue concentrations vary widely. Here, we identified inositol 1,4,5-trisphosphate (IP3) receptor type 3 as a regulator of axonal sensitivity to guidance cues in vitro and in vivo. Growth cones lacking the type 3 subunit are hypersensitive to nerve growth factor (NGF), an IP3-dependent attractive cue, and incapable of turning toward normal concentration ranges of NGF to which wild-type growth cones respond. This is due to globally, but not asymmetrically, activated Ca2+ signaling in the hypersensitive growth cones. Remarkably, lower NGF concentrations can polarize growth cones for turning if IP3 receptor type 3 is deficient. These data suggest a subtype-specific IP3 receptor function in sensitivity adjustment during axon navigation., Graphical Abstract, Highlights • IP3 receptor type 3 (IP3R3) controls axonal sensitivity to IP3-based guidance cues • IP3R3−/− growth cones are not attracted to NGF due to global Ca2+ responses • Lower NGF concentrations can polarize IP3R3−/− growth cones for attractive turning • NGF knockdown in vivo can revert abnormal trajectory of IP3R3−/− axons, Biological Sciences; Neuroscience; Molecular Neuroscience; Cellular Neuroscience

JAK2-V617F plays a key role in the pathogenesis of myeloproliferative neoplasm. However, its inhibitor ruxolitinib has shown limited clinical efficacies because of the ruxolitinib-persistent proliferation of JAK2-V617F-positive cells. We here demonstrate that the USP9X inhibitor WP1130 or EOAI3402143 (G9) inhibited proliferation and induced apoptosis more efficiently in cells dependent on JAK2-V617F than on cytokine-activated JAK2. WP1130 preferentially downregulated activated and autophosphorylated JAK2-V617F by enhancing its K63-linked polyubiquitination and inducing its aggresomal translocation to block downstream signaling. Furthermore, JAK2-V617F associated physically with USP9X in leukemic HEL cells. Induction of apoptosis by inhibition of USP9X was mediated through the intrinsic mitochondria-mediated pathway, synergistically enhanced by BH3 mimetics, prevented by overexpression of Bcl-xL, and required oxidative stress to activate stress-related MAP kinases p38 and JNK as well as DNA damage responses in HEL cells. Although autophosphorylated JAK2-V617F was resistant to WP1130 in the ruxolitinib-persistent HEL-R cells, these cells expressed Bcl-2 and Bcl-xL at lower levels and showed an increased sensitivity to WP1130 as well as BH3 mimetics as compared with ruxolitinib-naive HEL cells. Thus, USP9X represents a promising target along with anti-apoptotic Bcl-2 family members for novel therapeutic strategies against JAK2-V617F-positive myeloproliferative neoplasms, particularly under the ruxolitinib persistence conditions.

Background: Targeting apoptosis pathways in cancer has been extensively studied including the recent breakthrough therapy using venetoclax in acute myeloid leukemia (AML). However, certain therapy-resistant subsets of AML still pose a clinical challenge, leading us to investigate therapeutic strategies to bypass apoptosis pathways. Recently, non-apoptotic regulated cell death modes have attracted attention. Ferroptosis is a form of regulated cell death characterized by reactive oxygen species (ROS) induction and iron-dependent lipid peroxidation. Leukemia cells exhibit increased oxidative stress, which is further enhanced by iron overload, but only limited studies have explored therapeutic potential of targeting ferroptosis in AML. With mitochondria being the center for ROS production and iron metabolism containing 20-50% of cellular iron, mitochondrial (mito-) regulation of ferroptosis remains unclear. Incorporating our recent knowledge on mito-protease ClpP, hyperactivation of which selectively kills cancer cells through mito-proteolysis and oxidative stress (Ishizawa et al. 2019 Cancer Cell), we here aim to study the therapeutic targeting of the ferroptosis pathway and potential combinatorial targeting of mitochondria in AML. Results: We first utilized the TCGA dataset to analyze gene expression of key components in one of the major anti-ferroptosis pathways cysteine-glutathione-GPX4 axis. AML patients with higher mRNA expression of SLC7A11 (cystine importer), GCLM (a subunit of rate-limiting glutathione synthetase) or GPX4 have significantly shorter overall survival (p < 0.05), suggesting potential prognostic impact of this pathway. Here we focused on GPX4, the most downstream molecule that reduces lipid hydroperoxide to block ferroptosis. Although GPX4 is essential for embryonic development in mice, acquired depletion with conditional knock out was reported to have no significant effect on the number and function of hematopoietic stem cells, suggesting the tolerability of GPX4-targeted therapy. We demonstrate that GPX4 inhibition with sub-micromolar concentrations of ML210, a covalent GPX4 inhibitor with proteome-wide specificity, or with shRNA-mediated knockdown induces prominent cell death in various AML cell lines. The anti-leukemia effects were associated with lipid peroxidation and were almost completely abrogated by a lipophilic antioxidant Liproxstatin-1 (Lip1). The effect of ML210 was also blocked by the iron chelator deferoxamine, indicating ferroptosis. We also demonstrate that GPX4 knockdown increases ROS prominently in mitochondria before cell death is induced, suggesting that mitochondria is involved in the anti-leukemia effects. Meanwhile, GPX4 is among the top 15 sensitization hits in a previously published genome-wide CRISPR screening of leukemia cells treated with potent ClpP agonists ONC201 and ONC212 (Jacques et al. 2020 Genetics), indicating the protective function of GPX4 against mito-oxidative stress. We then found that genetic or pharmacologic hyperactivation of ClpP upregulated GPX4 protein expression predominantly in mitochondria in AML cells, collectively suggesting that upregulation of mito-GPX4 is cell-protective against ClpP-mediated cell death. Indeed, combinatorial ClpP hyperactivation enhanced the anti-leukemia effects of GPX4 inhibition through increased induction of mito-ROS and lipid peroxidation. We further demonstrate that the synergistic cell killing is inhibited by Lip1, indicating that the dual targeting of ClpP and GPX4 induces lipid peroxidation-mediated cell death. Furthermore, the inhibition of lipid peroxidation simultaneously resulted in over 80% reduction of mito-ROS, unexpectedly. This suggests that most of the increased mito-ROS by this combination is associated with lipid peroxidation. Mechanisms of induction of mitochondrial lipid peroxides jointly engaged by ClpP and mito-GPX4, as well as its involvement in the induced oxidative cell death, have yet to be explored. Conclusion: Our data suggests the potential involvement of mitochondrial lipid peroxidation in the anti-leukemia effects of GPX4 inhibition, along with its therapeutic potential in conjunction with mito-oxidative stress induction though instability in mito-proteome. Further investigations are in progress to assess the molecular mechanisms and the in vivo efficacy of the combinatorial treatment. Disclosures Andreeff: Oxford Biomedica UK: Research Funding; Aptose: Consultancy; Medicxi: Consultancy; Syndax: Consultancy; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Research Funding; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; AstraZeneca: Research Funding.

【Introduction】 Although Ruxolitinib (RUX), a JAK1/2-inhibitor, is an effective treatment option for primary myelofibrosis, tumor cells become resistant to this drug in many MPN patients, causing poor prognosis of MPN patients. Until now, various studies have elucidated the mechanisms of RUX-resistance in JAK2V617F-mutant MPN cells, including 1) increased JAK2 heterodimerization leading to sustained JAK2 activation, 2) JAK2 kinase domain mutations, and 3) JAK1/JAK3 activations. However, mechanisms of RUX-resistance in MPN cells with CALR mutations have not been fully characterized to date. In this study, we have clarified a mechanism of RUX-resistance in MPN tumor cells with CALR mutations. 【Materials and Methods】 At first, we have created several human cell lines with exogenous MPL expressions (exMPL) and CALR +1 frameshift mutations (CALR-fs) by introductions of V5-tagged MPL and/or FLAG-tagged CALR Del52/Ins5 expressing vectors or the CRISPR/Cas9 technology. We have confirmed that these cell lines had increased JAK/STAT signaling and respond to RUX-treatment. To establish RUX-resistant cell lines, we have cultured these cell lines with low-dose RUX (0.2μM), and gradually increased the concentrations of RUX by 0.1μM every week. We have successfully established RUX-resistant cells that proliferated in the presence of RUX at 0.8μM. Then, we characterized the RUX-resistant cells with CALR-fs/exMPL. To examine whether RUX-resistant cells shows the resistant-phenotype in vivo, we have subcutaneously implanted RUX-resistant cells as well as RUX-sensitive cells into immunocompromised mice. Three weeks after injections of the tumor cells, the mice were euthanized, and the subcutaneous tumors pathologically examined. MPL knockdown experiments showed that high levels of MPL were indispensable in the resistant cell lines. Co-immunoprecipitation assaies showed the interactions of mutant CALR and MPL proteins in RUX-resistant cells. To examine reversibility of RUX-resistance, RUX-resistant cells were cultured without RUX for three months. Finally, we examined pathological features of bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. 【Results】We have found that RUX-resistant cells had high MPL transcripts, overexpression of both immature and mature MPL, and JAK2. We also found that RUX-resistant cells had increased phosphorylations of JAK1, JAK2, JAK3 STAT5, MEK and ERK. In vivo assay using immunocompromised mice showed the immunohistochemical staining of MPL in the tumors from RUX-resistant cells showed high expression of MPL in the tumor cells. We also found that mature MPL proteins were more stable since proteasome-dependent degradation of mature MPL proteins was impaired in RUX-resistant cells. Knockdown of MPL of RUX-resistant cells by shRNAs decreased intensity of phosphorylations of JAK1, JAK2, STAT5, MEK and ERK, suggesting that the high expression of MPL leads to more potent signaling. Notably, when mutant-CALR proteins were immunoprecipitated, both immature and mature MPL proteins were co-immunoprecipitated: more MPL proteins were pulled down in RUX-resistant cells. In a reciprocal experiment, when MPL proteins were immunoprecipitated, mutant-CALR proteins were co-immunoprecipitated: more mutant-CALR proteins were pulled down in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells, suggesting that a high level of mutant-CALR/MPL complex contributed to RUX-resistance in these cells. When the resistant cells were cultured in the absence of RUX, RUX-resistance was reversed with reduction of MPL transcripts, mature MPL and JAK2 proteins and mutant-CALR/MPL complex. Immunohistochemical staining showed that MPL staining intensity of megakaryocytes of MF patients with CALR mutations were higher than those of patients with JAK2V617F or normal individuals. MPL expressions were higher after RUX-treatment in MF patients with both JAK2V617F mutations and CALR mutations. 【Discussion】 Overexpression of MPL is a common mechanism after RUX-treatment in MPN cells with both JAK1V617F mutations and CALR mutations. In RUX-resistant cells with CALR mutations, MPL overexpression leads to the high level of mutant-CALR/MPL complex, causing resistance to RUX. This novel mechanism could be a new therapeutic target to overcome RUX-resistance in MPN cells with CALR mutations. Disclosures Komatsu: Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding.

Cancer cells depend on aberrant antioxidant mechanisms to survive increased oxidative stress, which is particularly critical for cancer stem cell maintenance and its therapy resistance phenotype. Mitochondria is the primary site of oxygen consumption and reactive oxygen species (ROS) production, thus is essential for regulating cellular redox homeostasis. We have recently reported that hyperactivation of mitochondrial (mito-) protease CLPP by imipridones (ONC201/212) exerts cancer selective lethality through degradation of mito-proteins including respiratory chain complexes and induction of oxidative stress (Ishizawa et al., Cancer Cell 2019). However, the cell death mechanisms remain to be fully determined. We hypothesized that deregulation of mito-redox homeostasis induces a distinct mode of cell death in CLPP-hyperactivated cells and that concomitant inhibition of both mito- and cytosolic antioxidant mechanisms could enhance the anti-leukemia effects. We first tested if inhibition of glutathione (GSH) synthesis enhances the anti-leukemia effects by CLPP hyperactivation by using buthionine sulfoximine (BSO), a glutamate-cysteine ligase inhibitor. As hypothesized, BSO highly sensitized AML cells to genetic or pharmacologic hyperactivation of CLPP by overexpressing CLPP-Y118A (an activating mutation) or ONC201 treatment. We next investigated if a downstream target of GSH depletion is causing the synergy, and re-analysis of recently published CRISPR-knockout screens using leukemic cells treated with imipridones identified GPX4 as one of the 15 top-hit genes whose knockout sensitized cells to CLPP hyperactivation. GPX4 is a selenoprotein that is known for its GSH-dependent reduction of lipid hydroperoxides to protect cells from oxidative cell death including ferroptosis. We found that CLPP hyperactivation increased GPX4 protein levels in AML cells, prominently in mitochondria. Consistently, the GPX4 inhibitors ML210 and JKE1674 sensitized cells to ONC201 in multiple AML cell lines. Overexpression of the activating CLPP-Y118A was also synthetically lethal to GPX4 inhibition, confirming the on-target effect of ONC201. Mechanistically, GPX4 inhibition induced iron-dependent lipid peroxidation and AML cell killing, thus defining ferroptosis as the operational mechanism of cell death. Dual targeting of CLPP and GPX4 induced enhanced lipid peroxidation and mito-ROS but ferroptosis inhibitors only partially blocked cell death, indicating that other modes of oxidative cell death are also involved in the combinatorial synergism. Collectively, our data indicate therapeutic potential of targeting GPX4 and CLPP in AML and potential identification of a novel mode of cancer cell death. Investigations are ongoing to elucidate the underlying molecular mechanisms and to assess in vivo efficacy of the combinatorial treatment. Citation Format: Hiroki Akiyama, Ran Zhao, Lauren B. Ostermann, Jo Ishizawa, Michael Andreeff. Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML) [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 2017.

The t(3;21)(q26.2;q22) translocation is a rare chromosomal abnormality exhibited almost exclusively in therapy-related myelodysplastic syndrome/acute myeloid leukemia (t-MDS/AML) or in the blastic crisis phase of chronic myelogenous leukemia, which results in the fusion of the runt related transcription factor 1 (RUNX1, also called AML1) gene at 21q22 to the myelodysplasia syndrome 1 (MDS1)-ecotropic virus integration site 1 (EVI1) complex locus (MECOM) at 3q26.2, generating various fusion transcripts, including AML1/MDS1/EVI1 (AME). The present study examined the case of an 84-year-old Japanese woman who developed t-MDS/AML with t(3;21)(q26.2;q22) subsequent to receiving low-dose methotrexate (MTX) treatment for rheumatoid arthritis. Following treatment with MTX for 6 years, the patient developed anemia and neutropenia, and MTX was discontinued. A total of 3 years later, the patient was diagnosed with MDS with t(3;21)(q26.2;q22) and del (5q), which progressed rapidly to AML within 3 months. The patients was subsequently treated with azacitidine and cytarabine chemotherapy, but succumbed to the disease 6 months after diagnosis. Sequencing analysis of the nested reverse transcription-PCR products from the leukemic cells revealed the expression of two types of alternatively-spliced AME transcripts with or without RUNX1 exon 6 sequences. Western blot analysis of the leukemic cells of the patient additionally revealed that the corresponding AME fusion protein products were expressed at high levels, and that these cells also prominently expressed CCAAT/enhancer-binding protein α, the repression of which has been reported to be involved in leukemogenesis mediated by AME. To the best of our knowledge, the case discussed in the present study represents the first report of MDS/AML with t(3;21)(q26.2;q22) developing following low-dose MTX therapy for rheumatoid arthritis. Nonetheless, the clinical and molecular features of the patient in the present study were representative of those patients who typically develop this disease following exposure to chemotherapy or radiotherapy for primary malignancy, which implicates MTX in the pathogenesis of t-MDS/AML. Moreover, we confirmed the expression of two AME fusion proteins for the first time in primary leukemic cells and analyzed several cellular factors implicated in AME-mediated leukemogenesis to gain some insight into its molecular mechanisms.

【 Introduction 】 CAR-T cell therapy has shown excellent therapeutic effects against some malignancies including refractory B cell lymphoma or leukemia. This adoptive T cell transfer therapy is an attractive methodology; however, there are several disadvantages to be overcome. Current CAR-T cell therapy targets single cell-surface molecule, which leads to damage of normal cells expressing the target protein. Such "On-target / off-tumor" effect is one of the major adverse effects. Improvement of target-cell-specificity is needed to avoid this serious adverse effect and to expand target diseases of this therapy. Here, we show the protease-mediated "Double-Arm" CAR-T cell system, which improved the specificity of CAR-T cell therapy by recognizing two distinct cell-surface proteins. We designed two types of CARs: "Effector CAR" and "Scissors CAR". The "Effector CAR" is constituted of a single chain Fv fragment (scFv) targeting a cell-surface protein (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition polypeptide sequence, and a functional domain of CD3-zeta. The "scissors CAR" is constituted of a recognition portion targeting another protein (protein Y) and HIVPR. The HIVPR induces cleavage of the recognition polypeptide sequence in the effector CAR leading to inactivation of the effector CAR when the CAR-T cells contact with cells expressing both proteins X and Y. 【 Material and Methods 】 For proof of principle, we first constructed "anti-CD19 mCherry CAR" harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition polypeptide sequence. Also, we constructed "anti-CD19 scissors CAR", low affinity "anti-HER2 (4D5-3) scissors CAR" and high affinity "anti-HER2 (4D5-8) scissors CAR". To analyze the target-cell-dependent cleavage of mCherry CAR, 293T cells expressing these CARs were co-cultured with target cells, including K562 (CD19-, HER2-), Raji (CD19+, HER2+), or SK-BR-3 (CD19-, HER2+). To obtain target cells expressing both CD19 and HER2, Raji and SK-BR-3 cells were molecularly manipulated. (1) To evaluate efficiency of this system, after co-cultivation of CAR-transduced 293T and the target cells (K562, Raji, SK-BR-3), the localization of mCherry was examined under the microscopy and Western blotting. (2) To assess the T cell activation, we constructed "anti-CD19 effector CAR" and established Jurkat cells expressing both the "effector CAR" and the "anti-HER2 scissors CAR". These cells were co-cultured with wild type or engineered Raji or SK-BR-3 cells. T cell activation was analyzed with flowcytometric analysis. (3) To assess the CAR activity of this system, we transduced these CARs to primary T cells from healthy donors. After co-cultivation with the target cells, we measured target-specific cytotoxic activity. 【 Results 】 (1) Transduced "anti-CD19 mCherry CAR" was detected as a membrane-bound protein in 293T cells. Co-cultivation of 293T cells expressing both the "anti-CD19 mCherry CAR" and "anti-HER2 scissors CAR" with engineered Raji cells expressing both CD19 and HER2 induced cleavage of the recognition site and translocation of the mCherry from the membrane to the cytoplasm. These results suggested that this system would regulate activities of CAR-T cell through HIVPR-mediated cleavage of the "effector CAR" in vitro. (2) Jurkat cells expressing "anti-CD19 effector CAR" were activated through a target-cell-dependent manner. Furthermore, "anti-HER2 scissors CAR" attenuated T cell activation driven by "anti-CD19 effector CAR" when Jurkat cells expressing both the "anti-CD19 effector CAR" and the "anti-HER2 scissors CAR" contacted with target-cells expressing both CD19 and HER2. In addition, high affinity 4D5-8 scissors CAR showed more potent attenuation than the low affinity 4D5-3 scissors CAR. (3) Primary human T cells expressing "anti-CD19 effector CAR" showed CD19-expressing target-cell-specific cytotoxic activity. We also demonstrated that "Double Arm" primary human CAR-T cells showed tumor-cell-specificity as seen in cell-line models described above. 【Discussion】 Our "Double-Arm" CAR-T cell system has improved tumor-cell-specificity even in primary human T-cells as we expected. This system would attenuate the adverse effects of clinical CAR-T cell therapies. Disclosures No relevant conflicts of interest to declare.

// Yoshihiro Umezawa 1 , Tetsuya Kurosu 1 , Hiroki Akiyama 1 , Nang Wu 1 , Ayako Nogami 1 , Toshikage Nagao 1 , Osamu Miura 1 1 Department of Hematology, Graduate School of Medical and Dental Sciences, and Graduate School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan Correspondence to: Osamu Miura, email: miura.hema@tmd.ac.jp Keywords: Chk1, p53, BCR/ABL, Jak2, chemotherapeutics Received: February 17, 2016 Accepted: May 20, 2016 Published: June 06, 2016 ABSTRACT DNA-damaging chemotherapeutic agents activate apoptotic pathways in cancer cells. However, they also activate checkpoint mechanisms mainly involving Chk1 and p53 to arrest cell cycle progression, thus abbreviating their cytotoxic effects. We previously found that aberrant tyrosine kinases involved in leukemogenesis, such as BCR/ABL and Jak2-V617F, as well as Jak2 activated by hematopoietic cytokines enhance Chk1-mediated G2/M arrest through the PI3K/Akt/GSK3 pathway to confer resistance to chemotherapeutic agents, which was prevented by inhibition of these kinases or the downstream PI3K/Akt pathway. However, the possible involvement of p53 in regulation of Chk1-mediated G2/M checkpoint has remained to be elucidated. We demonstrate here that a dominant negative mutant of p53, p53-DD, increases Chk1-mediated G2/M checkpoint activation induced by chemotherapeutics and protects it from down regulation by inhibition of Jak2, BCR/ABL, or the PI3K/Akt pathway in hematopoietic model cell lines 32D and BaF3 or their transformants by BCR/ABL. Consistent with this, the p53 activator nutlin-3 synergistically induced apoptosis with chemotherapeutics by inhibiting Chk1-mediated G2/M arrest in these cells, including cells transformed by the T315I mutant of BCR/ABL resistant to various kinase inhibitors in clinical use. Further studies suggest that p53 may inhibit the Chk1 pathway by its transcription-dependent function and through mechanisms involving the proteasomal system, but not the PI3K/Akt/GSK3 pathway. The present study may shed a new light on molecular mechanisms for the therapy resistance of p53-mutated hematological malignancies and would provide valuable information for the development of novel therapeutic strategies against these diseases with dismal prognosis.

Graded distribution of intracellular second messengers, such as Ca2+and cyclic nucleotides, mediates directional cell migration, including axon navigational responses to extracellular guidance cues, in the developing nervous system. Elevated concentrations of cAMP or cGMP on one side of the neuronal growth cone induce its attractive or repulsive turning, respectively. Although effector processes downstream of Ca2+have been extensively studied, very little is known about the mechanisms that enable cyclic nucleotides to steer migrating cells. Here, we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance via modulating microtubule dynamics and membrane organelle transport. In embryonic chick dorsal root ganglion neurons in culture, contact of an extending microtubule with the growth cone leading edge induces localized membrane protrusion at the site of microtubule contact. Such a contact-induced protrusion requires exocytosis of vesicle-associated membrane protein 7 (VAMP7)-positive vesicles that have been transported centrifugally along the microtubule. We found that the two cyclic nucleotides counteractively regulate the frequency of microtubule contacts and targeted delivery of VAMP7 vesicles: cAMP stimulates and cGMP inhibits these events, thereby steering the growth cone in the opposite directions. By contrast, Ca2+signals elicit no detectable change in either microtubule contacts or VAMP7 vesicle delivery during Ca2+-induced growth cone turning. Our findings clearly demonstrate growth cone steering machinery downstream of cyclic nucleotide signaling and highlight a crucial role of dynamic microtubules in leading-edge protrusion for cell chemotaxis.SIGNIFICANCE STATEMENTDeveloping neurons can extend long axons toward their postsynaptic targets. The tip of each axon, called the growth cone, recognizes extracellular guidance cues and navigates the axon along the correct path. Here we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance through localized regulation of microtubule dynamics and resulting recruitment of specific populations of membrane vesicles to the growth cone's leading edge. Remarkably, cAMP stimulates microtubule growth and membrane protrusion, whereas cGMP promotes microtubule retraction and membrane senescence, explaining the opposite directional polarities of growth cone turning induced by these cyclic nucleotides. This study reveals a novel microtubule-based mechanism through which cyclic nucleotides polarize the growth cone steering machinery for bidirectional axon guidance.

Neuronal networks in our brain and body are fundamental for sensation, behavior, and all other functions based on neuronal activity. To construct precisely wired networks, the development of neurons is tightly controlled. During development, neurons must alter their shape so as to be able to migrate toward the destination from their origin and to make contact, called a synapse, with a defined target a certain distance away from them. Like the skeleton in our body, neurons have a cytoskeleton to maintain their shape. The cytoskeleton not only supports this shape, but also helps regulate the motility of neurons; and in this regard, the cytoskeleton is comparable to muscle. Therefore, orchestrating the reorganization of the cytoskeleton is of great importance in allowing extensive changes in neuronal morphology during development. In this review, we highlight the crucial roles of the cytoskeleton and associated proteins in each phase of neuronal development, including neurogenesis, neuronal migration, neurite genesis and growth, and synapse formation.

Bi 2 Sr 2 CaCu 2 O 8+δ (Bi2212) mesas are promising candidates for THz oscillators, which can fill the frequency range around “THz gap”. However, it is known that Bi2212 mesas show self-heating effects (hot spots) when the current is passed along the c -axis due to the low thermal conductivity in this direction. Although several previous studies reported the relation of the hot spot and THz emission, consistent answer has not been obtained yet. In order to address this issue, imaging of temperature distributions on Bi2212 mesas is expected to be very effective. Here, we set up fluorescent thermal imaging (FTI) method for visualizing the surface temperature distribution on the Bi2212 mesa. We have succeeded in observing hot spots in the Bi2212 mesa with high spatial resolution.

[Introduction] CAR-T cell therapy is an attractive methodology in the field of cancer immunotherapy. The anti-CD19 CAR-T cell therapy shows excellent therapeutic effects against refractory B-cell malignancies. However, the CAR-T cells attack not only target-tumor-cells, but also normal tissues expressing the target molecule, for example, normal B-cells with CD19. For the CAR-T cell therapy, such “on-target / off-tumor” effect is one of major obstacles to be overcome. Here, we show the protease-mediated regulatory CAR-T cell system, which improved the target-specificity by recognizing two distinct antigens. We designed two types of CAR: "effector CAR" and "scissors CAR". The “effector CAR” is constituted of a single chain Fv fragment (scFv) targeting an antigen (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition site, and a functional domain of CD3-ζ. The “scissors CAR” is constituted of a scFv targeting another antigen (protein Y) and HIVPR. The “scissors CAR” induces cleavage of the “effector CAR” leading to inactivation when the CAR-T cells contact with cells expressing both proteins X and Y. [Material and Methods] For proof of principle, we first constructed the anti-CD19 “mCherry CAR” harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition site. Also, we constructed the anti-HER2 “scissors CAR”. To analyze the target-cell dependent cleavage of “mCherry CAR”, 293T cells expressing the CARs were co-cultured with target-cells, including Raji (CD19+, HER2-) and engineered SK-BR-3 (CD19+, HER2+). Locations of mCherry were analyzed under the microscopy and by Western blotting. Next, to assess T-cell activation, we established Jurkat cells expressing both the anti-CD19 “effector CAR” and the anti-HER2 “scissors CAR”. These cells were co-cultured with the target-cells described above. T-cell activation was analyzed by both flowcytometric analysis and measurement of IL-2 mRNA expression. [Results] Transduced anti-CD19 “mCherry CAR” was detected as a membranous protein in 293T cell. Co-cultivation of 293T cell expressing both the anti-CD19 “mCherry CAR” and the anti-HER2 “scissors CAR” with Raji (CD19+, HER2-) did not change the localization of mCherry. However, engineered SK-BR-3 (CD19+, HER2+) induced cleavage of the recognition site and translocation of the mCherry from the membrane to cytoplasm. Furthermore, the anti-HER2 “scissors CAR” attenuated T-cell activation driven by the anti-CD19 “effector CAR” when Jurkat cells expressing both the CARs contacted with the target-cells expressing both CD19 and HER2. [Discussion] This novel protease-mediated CAR-T system recognized expression pattern of target molecules and regulated T-cell activity. Our CAR-T system would attenuate the adverse effects and contribute to expansion in application of CAR-T cell therapy. Citation Format: Satoru Aoyama, Shunichiro Yasuda, Daisuke Watanabe, Hiroki Akiyama, Keigo Okada, Yoshihiro Umezawa, Ayako Nogami, Osamu Miura, Norihiko Kawamata. A protease-mediated regulatory chimeric antigen receptor (CAR): “Scissors” CAR improves target-specificity through regulation of T-cell activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2167.

FLT3-ITD and FLT3-TKD are the most frequent mutations in acute myeloid leukemia (AML) with the former associated with a poor prognosis. Here we show that inhibition of the deubiquitinase USP9X by its inhibitor WP1130 or EOAI3402143 (G9) induces apoptosis preferentially in cells transformed by these mutant kinases, including FLT3-ITD-positive AML cell line MV4-11 and primary AML cells. Mechanistically, WP1130 induced aggresomal translocation of the mutant kinases, particularly FLT3-ITD in its activated and autophosphorylated conformation, to block the downstream signaling events, which was aggravated by knock down of USP9X. Moreover, USP9X physically associated with FLT3-ITD to inhibit its K63-linked polyubiquitination, while FLT3-ITD induced tyrosine phosphorylation and degradation of USP9X through the ubiquitin/proteasome pathway. WP1130 or G9 also induced oxidative stress to stimulate stress-related MAP kinase pathways and DNA damage responses to activate in cooperation with inhibition of FLT3-ITD signaling the intrinsic mitochondria-mediated apoptotic pathway, which was synergistically enhanced by BH3 mimetics and prevented by overexpression of Bcl-xL or Mcl-1. Thus, USP9X represents a promising target for novel therapies against therapy-resistant FLT3-ITD-positive AML.

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously found that FLT3-ITD activates the mTORC1/S6K/4EBP1 pathway cooperatively through the STAT5/PIM and PI3K/AKT pathways to promote proliferation and survival by enhancing the eIF4F complex formation required for cap-dependent translation. Here, we show that, in contrast to BCR/ABL causing Ph-positive leukemias, FLT3-ITD distinctively activates the serine/threonine kinases RSK1/2 through activation of the MEK/ERK pathway and PDK1 to transduce signals required for FLT3-ITD-dependent, but not BCR/ABL-dependent, proliferation and survival of various cells, including MV4-11. Activation of the MEK/ERK pathway by FLT3-ITD and its negative feedback regulation by RSK were mediated by Gab2/SHP2 interaction. RSK1 phosphorylated S6RP on S235/S236, TSC2 on S1798, and eIF4B on S422 and, in cooperation with PIM, on S406, thus activating the mTORC1/S6K/4EBP1 pathway and eIF4B cooperatively with PIM. RSK1 also phosphorylated Bad on S75 and downregulated BIM-EL in cooperation with ERK. Furthermore, inhibition of RSK1 increased sensitivities to BH3 mimetics inhibiting Mcl-1 or Bcl-2 and induced activation of Bax, leading to apoptosis, as well as inhibition of proliferation synergistically with inhibition of PIM or PI3K. Thus, RSK1 represents a promising target, particularly in combination with PIM or PI3K, as well as anti-apoptotic Bcl-2 family members, for novel therapeutic strategies against therapy-resistant FLT3-ITD-positive AML.

【Introduction】 Although Ruxolitinib (Rux), a JAK1/2-inhibitor, is an effective treatment option for primary myelofibrosis, tumor cells become resistant to this drug in many MPN patients. Currently, Rux-resistance is a major problem and contributes to poor prognosis of MPN patients. Mechanisms of Rux-resistance in MPN cells with JAK2V617F mutations have already been reported. On the other hand, MPN cells with CALR mutations resistant to Rux-treatment have not been fully characterized yet. In this study, we have clarified a mechanism of Rux-resistance in MPN tumor cells with CALR mutations in both cell lines and clinical samples. 【Materials and Methods】To establish human cell lines with CALR mutations, we have introduced deletion of 46-nucleotides generating the CALR frame-shift mutation (del46), one of the type1-like mutations, to UT-7/EPO by the CRISPR/Cas9 system. Then, we introduced exogenous MPL to UT-7/EPO with the CALR mutations by transfection. We confirmed that UT-7/EPO with CALR frame-shift mutations (CALR-fs) and exogenous MPL (exMPL) activate STAT5 and showed cytokine-independent cell growth. To establish Rux-resistant cell lines, we have cultured these cell lines with low-dose Rux (0.2μM), and gradually increased the concentrations of Rux by 0.1μM every week. Cells were considered as resistant when the half-maximal inhibitory concentrations (IC50) of the cells was double the IC50 of the parental cells. We have successfully established Rux-resistant cells that proliferated in the presence of Rux at 0.8μM. Then, we characterized the Rux-resistant cells with CALR-fs/exMPL. Next, reversibility of Rux-resistance, the Rux-resistant cells was examined. To examine whether Rux-resistant cells shows the resistant-phenotype in vivo, we have subcutaneously implanted Rux-resistant cells as well as Rux-sensitive cells into immunocompromised mice and treated the animals with Rux. Three weeks after injections of the tumor cells, the mice were euthanized, and the subcutaneous tumors were excised and weighted. Finally, we examined pathological features of bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. 【Results】We found that Rux-resistant cells had overexpressions of both MPL and JAK2, and increased phosphorylations of both JAK2 and STAT5. We found that high levels of MPL transcripts in the resistant cells. Cycloheximide treatment assay showed that mature MPL proteins were more stable in Rux-resistant cells. Then, we examined mechanisms of stability of mature MPL proteins in Rux-resistant cells. Our data suggested that the proteasome/system degrading mature MPL was attenuated in Rux-resistant cells. When Rux-resistant cells were cultured in the absence of Rux, Rux-resistance was reversed with reduction of mature MPL and JAK2. In vivo assay using immunocompromised mice showed the Rux resistant cells were not sufficiently suppressed by Rux-treatment. Since our data using the cell lines suggested that high MPL expression causes resistance to Rux in MPN cells with CALR mutations, we examined levels of MPL proteins in bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. Immunohistochemical staining of MPL showed that the megakaryocytes of MPN patients with high-risk and/or resistant to ruxolitinib treatment expressed higher MPL. 【Discussion】 MPL overexpression is one of mechanisms of MPN cells with CALR mutations to gain resistance to Rux. This mechanism may be a key element to overcome Rux-resistance in this fatal disease. Disclosures Komatsu: Novartis K.K: Speakers Bureau; Wako Pure Chemical Industries, Ltd.: Research Funding; Fuso Pharmaceutical Industries, Ltd.: Research Funding; Takeda Pharmaceutical Company Limited: Research Funding, Speakers Bureau; Pharma Essentia: Research Funding, Speakers Bureau.

【 Introduction 】 CAR-T cell therapy is an attractive methodology in the field of cancer immunotherapy. Anti-CD19 CAR is used for treating refractory B cell malignancies and shows excellent therapeutic effects. However, there are several disadvantages to be overcome in this therapy. "On-target / off-tumor" effect is one of such adverse effects. Current CAR-T cell therapy targets single cell-surface molecule, which leads to damage of normal cells expressing the target protein. Improvement of target-cell-specificity is one of important issues in CAR-T cell therapy to avoid this serious adverse effect. Here, we show the protease-mediated "Double-Arm" CAR-T cell system (Figure), which improved the specificity of CAR-T cell therapy by recognizing two distinct cell-surface proteins. We designed two types of CAR: "Effector CAR" and "Scissors CAR" (Figure, left panel). The "effector CAR" is constituted of a single chain Fv fragment (scFv) targeting a cell-surface protein (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition polypeptide sequence, and a functional domain of CD3-zeta. The "scissors CAR" is constituted of a recognition portion targeting another protein (protein Y) and HIVPR. The HIVPR induces cleavage of the recognition polypeptide sequence in the effector CAR leading to inactivation of the effector CAR when the CAR-T cells contact with cells expressing both protein X and Y (Figure, right panel). 【 Material and Methods 】 For proof of principle, we first constructed "anti-CD19 mCherry CAR" harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition polypeptide sequence. Also, we constructed "anti-CD19 scissors CAR" and "anti-HER2 scissors CAR". To analyze the target-cell-dependent cleavage of mCherry CAR, 293T cells expressing these CARs were co-cultured with target cells, including K562 (CD19-, HER2-), Raji (CD19+, HER2+), or SK-BR-3 (CD19-, HER2+). To obtain target cells expressing both CD19 and HER2, Raji and SK-BR-3 cells were molecularly manipulated. (1) To evaluate efficiency of this system, after co-cultivation of CAR-transduced 293T cell and target cells (K562, Raji, SK-BR-3), the localization of mCherry was examined under the microscopy and Western blotting. (2) To assess the T cell activation, we constructed "anti-CD19 effector CAR" and established Jurkat cells expressing both the "effector CAR" and the "anti-HER2 scissors CAR". These cells were co-cultured with wild type or engineered Raji or SK-BR-3 cells. T cell activation was analyzed with flowcytometric analysis and IL-2 mRNA expression measured with qRT-PCR. 【 Results 】 (1) Transduced "anti-CD19 mCherry CAR" was detected as a membrane-bound protein in 293T cell. Co-cultivation of 293T cell expressing both the "anti-CD19 mCherry CAR" and "anti-HER2 scissors CAR" with engineered Raji cells expressing both CD19 and HER2 induced cleavage of the recognition site and translocation of the mCherry from the membrane to cytoplasm. In addition, the cleavage was inhibited by a HIVPR inhibitor, Saquinavir. These results suggested that this novel system would regulate CAR-T cell activities through HIVPR-mediated cleavage of the "effector CAR" in vitro. (2) Jurkat cells expressing "anti-CD19 effector CAR" were activated through the target-cell-dependent manner. In addition, "anti-HER2 scissors CAR" attenuated T cell activation driven by "anti-CD19 effector CAR" when Jurkat cells expressing both the "anti-CD19 effector CAR" and the "anti-HER2 scissors CAR" contacted with the target cells expressing both CD19 and HER2. 【Discussion】 This is a novel protease-mediated controllable CAR system. The "scissors CAR" regulated activity of CAR-T cells depending on expression pattern of target molecules on the target cells. Our "Double-Arm" CAR-T cell system (Figure) would improve target specificity. It would attenuate the adverse effects and contribute to expansion in application of CAR-T cell therapy other than B cell malignancies. Figure Disclosures No relevant conflicts of interest to declare.

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously reported that FLT3-ITD activates the mTORC1/S6K/4EBP1 pathway cooperatively through the STAT5/PIM and PI3K/Akt pathways in AML cells, thus promoting proliferation and survival as well as therapy resistance at least partly by enhancing the eIF4F complex-mediated cap-dependent translation of growth- and survival-related mRNAs. The RSK family serine/threonine protein kinases, including the predominant isoforms expressed in AML RSK1 and RSK2, posses the N- and C-terminal kinase domains (NTKD and CTKD); CTKD is activated by Erk and recruits PDK1 to activate NTKD, which phosphorylates downstream substrates to promote cell growth and survival through regulation of translation as well as transcription. Although RSK2 was reported to be activated by FLT3-ITD or upregulated through PIM and to be required for leukemogenesis by FLT3-ITD but not by BCR/ABL, these studies examined only activation of CTKD, used mainly the CTKD inhibitor FMK, and did not adequately examine the downstream signaling mechanisms from RSK. Thus, we examined the significance of RSK in FLT3-ITD AML and the underlying molecular mechanisms. The RSK NTKD inhibitors LJI308 and LJH685 as well as FMK distinctively inhibited FLT3-ITD-dependent proliferation of the AML cell line MV4-11 and model hematopoietic cell lines, whereas BCR/ABL-dependent proliferation of various cells were resistant. Furthermore, inhibition of FLT3-ITD by quizartinib or gilteritinib, but not that of BCR/ABL by imatinib, prominently inhibited RSK1 and RSK2 NTKDs, as examined by the activation-specific phosphorylation in their T-loop sites, associated with inhibition of the MEK/Erk pathway and PDK1 in MV4-11. Inhibition of FLT3-ITD also reduced expression of RSK1 but not RSK2, which, however, was not mimicked by inhibition of PIM. Intriguingly, FMK or LJI308 inhibited FLT3-ITD and phosphorylation of its substrate STAT5 in MV4-11 cells as well as in primary AML cells expressing FLT3-ITD. By using the most specific RSK inhibitor LJH685 as well as sh-RNA-mediated knock down, CRISPR/CAS9-mediated knockout, or overexpression of RSK1 or RSK2 in MV4-11 cells, we found that mainly RSK1 but not RSK2 phosphorylates not only S6RP on S235/236 but also TSC2 on S1798 to upregulate the mTORC1/S6K/4EBP1 pathway. RSK1 also phosphorylated eIF4B on S422 and, in cooperation with PIM, on S408, which is required for efficient cap-dependent translation by the eIF4F complex. Furthermore, RSK1 phosphorylated Bad on S112, which is instrumental for dissociation from anti-apoptotic proteins Bcl-2 and Bcl-xL. On the other hand, inhibition of RSK or Erk by LJH685 or SCH772984, respectively, increased the expression level of BimEL in the S65-phosphorylated or unphosphorylated form, respectively, which is consistent with the idea that RSK phosphorylates BimEL after its phosphorylation on S65 by Erk to induce proteasomal degradation of this potent pro-apoptotic protein. Interestingly, inhibition of RSK upregulated the MEK/Erk pathway, which correlated with increased binding of SHP2 with Gab2 phosphorylated by FLT3-ITD and was prevented by the SHP2 inhibitor IIB-08, thus revealing the negative feed back mechanisms involving the Gab2/SHP2 complex. LJH685 or RSK1 knockout reduced viable cell numbers of MV4-11 as well as FLT3-ITD-positive primary AML cells synergistically with the PIM inhibitor AZD-1208 or the PI3K inhibitor GDC-0941 at least partly by inducing apoptosis, which was prevented by overexpression of Bcl-xL or Mcl-1. Knock down of mTOR in MV4-11 cells also enhanced apoptosis induced by inhibition of PIM or PI3K. Moreover, LJH685 sensitized MV4-11 significantly to BH3 mimetics, such as venetoclax or A-1210477, which inhibits Bcl-2 or Mcl-1, respectively. Our findings reveal that FLT3-ITD but not BCR/ABL activates RSK1 as well as RSK2 and upregulates RSK1 expression to promote proliferation and survival of leukemic cells through upregulation of the mTORC1 pathway and modification of eIF4B in collaboration with the PI3K/Akt and STAT5/PIM pathways and additionally through inhibitory modification of the proapoptotic proteins Bad and Bim (Figure). Thus, RSK1 represents a promising molecular target particularly in combination with PIM or PI3K for novel therapeutic strategies against therapy-resistant FLT3-ITD-positive AML. Disclosures No relevant conflicts of interest to declare.

A 40-year-old woman developed therapy-related acute myeloid leukemia (t-AML) with inv(16)(p13.1q22) and a rare type D form of core-binding factor β-subunit gene-myosin heavy chain 11 gene (CBFB-MYH11) fusion transcript approximately 2.5 years after receiving chemoradiotherapy for uterine cervical cancer. t-AML with inv(16)(p13.1q22) and rare non-type A CBFB-MYH11 typically develops after exposure to a topoisomerase II inhibitor, with a short period of latency of one to five years. As the patient had no history of exposure to topoisomerase II inhibitors, among her previously used chemotherapeutics, the topoisomerase I inhibitor, irinotecan, was speculated to be the most plausible cause of t-AML in this case. The present case suggests that irinotecan may cause t-AML resembling that associated with topoisomerase II inhibitors.

The prognosis of acute promyelocytic leukemia (APL) has become the most favorable among all acute myeloid leukemias, due to the efficacy of treatment with all-trans retinoic acid (ATRA). ATRA combined with anthracycline-based chemotherapy has significantly improved the long-term outcome for low-to-intermediate-risk APL patients; thus, the efficacy of maintenance therapy for patients achieving molecular complete remission (MCR) following consolidation therapy has become debatable. To evaluate the efficacy of maintenance therapy, we conducted a retrospective analysis of 11 consecutive patients with low-to-intermediate-risk APL who received induction and consolidation therapy with ATRA and anthracyclines according to the PETHEMA LPA protocols at our hospital between January, 2001 and March, 2013. All the patients achieved MCR following consolidation therapy. Of these patients, 7 were followed without maintenance therapy, including 2 patients who discontinued maintenance therapy within 2 months. With a median follow-up of 85 months, the overall survival for all the patients was 100%, while the disease-free survival estimate at 5 years with and without maintenance therapy was 100 and 85.7%, respectively; the difference was not statistically significant (P=0.45). Two patients treated with maintenance therapy later developed secondary primary malignancy. Thus, even without maintenance therapy, ATRA combined with anthracyclines exhibited significant efficacy in low-to-intermediate-risk APL patients, suggesting that maintenance therapy, which is associated with adverse events, may be dispensable for patients achieving MCR following adequate consolidation therapy.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell adhesion protein involved in the regulation of cell adhesion and migration. Interestingly, several PECAM-1-deficient hematopoietic cells exhibit impaired chemotactic responses to stromal cell-derived factor 1 (SDF-1), a chemokine essential for B lymphopoiesis and bone marrow myelopoiesis. However, whether PECAM-1 is involved in SDF-1-regulated chemotaxis is unknown. We report here that SDF-1 induces tyrosine phosphorylation of PECAM-1 at its immunoreceptor tyrosine-based inhibition motifs in several hematopoietic cell lines via the Src family kinase Lyn, Bruton's tyrosine kinase, and JAK2 and that inhibition of these kinases reduced chemotaxis. Overexpression and knockdown of PECAM-1 enhanced and down-regulated, respectively, SDF-1-induced Gαi-dependent activation of the PI3K/Akt/mTORC1 pathway and small GTPase Rap1 in hematopoietic 32Dcl3 cells, and these changes in activation correlated with chemotaxis. Furthermore, pharmacological or genetic inhibition of the PI3K/Akt/mTORC1 pathway or Rap1, respectively, revealed that these pathways are independently activated and required for SDF-1-induced chemotaxis. When coexpressed in 293T cells, PECAM-1 physically associated with the SDF-1 receptor CXCR4. Moreover, PECAM-1 overexpression and knockdown reduced and enhanced SDF-1-induced endocytosis of CXCR4, respectively. Furthermore, when expressed in 32Dcl3 cells, an endocytosis-defective CXCR4 mutant, CXCR4-S324A/S325A, could activate the PI3K/Akt/mTORC1 pathway as well as Rap1 and induce chemotaxis in a manner similar to PECAM-1 overexpression. These findings suggest that PECAM-1 enhances SDF-1-induced chemotaxis by augmenting and prolonging activation of the PI3K/Akt/mTORC1 pathway and Rap1 and that PECAM-1, at least partly, exerts its activity by inhibiting SDF-1-induced internalization of CXCR4.

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously reported that activation of STAT5 confers resistance to PI3K/Akt inhibitors on the FLT3-ITD-positive AML cell line MV4-11 and 32D cells driven by FLT3-ITD (32D/ITD) but not by FLT3 mutated in the tyrosine kinase domain (32D/TKD). Here, we report the involvement of Pim kinases expressed through STAT5 activation in acquisition of this resistance. The specific pan-Pim kinase inhibitor AZD1208 as well as PIM447 in combination with the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 cooperatively downregulated the mTORC1/4EBP1 pathway, formation of the eIF4E/eIF4G complex, and Mcl-1 expression leading to activation of Bak and Bax to induce caspase-dependent apoptosis synergistically in these cells. These cooperative effects were enhanced or inhibited by knock down of mTOR or expression of its activated mutant, respectively. Overexpression of Mcl-1 conferred the resistance on 32D/ITD cells to combined inhibition of the PI3K/Akt pathway and Pim kinases, while the Mcl-1-specific BH3 mimetic A-1210477 conquered the resistance of MV4-11 cells to GDC-0941. Furthermore, overexpression of Pim-1 in 32D/TKD enhanced the mTORC1/Mcl-1 pathway and partially protected it from the PI3K/Akt inhibitors or the FLT3 inhibitor gilteritinib to confer the resistance to PI3K/Akt inhibitors. Finally, AZD1208 and GDC-0941 cooperatively inhibited the mTORC1/Mcl-1 pathway and reduced viable cell numbers of primary AML cells from some FLT3-ITD positive cases. Thus, Pim kinases may protect the mTORC1/4EBP1/Mcl-1 pathway to confer the resistance to the PI3K/Akt inhibitors on FLT3-ITD cells and represent promising therapeutic targets.

Covalent conjugation of small ubiquitin-like modifiers (SUMOs) or SUMOylation is a reversible post-translational modification that regulates the stability and function of target proteins. SUMOs are removed from substrate proteins by sentrin/SUMO-specific proteases (SENPs). Numerous studies have implicated SUMOylation in various physiological and pathological processes in neurons. To understand the functional roles of SUMOylation, it is necessary to determine the distribution of enzymes regulating SUMO conjugation and deconjugation; yet, the localization of SENPs has not been described in detail in intact brain tissue. Here, we report the distribution and subcellular localization of SENP3 and 5 in the adult murine brain. Immunohistochemical analyses revealed the ubiquitous distribution of both SENPs across different brain regions. Within individual cells, SENP3 was confined to the nucleus, consistent with the conventional view that SENPs regulate nuclear events. In contrast, SENP5 was detected in the neuropil but not in cell bodies. Moreover, strong SENP5 immunoreactivity was observed in regions with high numbers of synapses such as the cerebellar glomeruli, suggesting that SENP5 localizes to pre- and/or postsynaptic structures. We performed double immunolabeling in cultured neurons and found that SENP5 co-localized with pre- and post-synaptic markers, as well as a mitochondrial marker. Immunoelectron microscopy confirmed this finding and revealed that SENP5 was localized to presynaptic terminals, postsynaptic spines, and mitochondria in axon terminals. These findings advance the current understanding of the functional roles of SUMOylation in neurons, especially in synaptic regulation, and have implications for future therapeutic strategies in neurodegenerative disorders mediated by mitochondrial dysfunction.

【Introduction 】 Myeloproliferative neoplasm (MPN) is a heterogeneous group of disorders, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). It is known that mutations of JAK2, CALR, or MPL gene are causative alternations of MPNs. However, 10-15% of MPNs have no mutations in any of these three genes, so-called "triple negative" (TN) MPN. It remains unclear that what genetic alternations cause TN-MPN. Cell lines derived from clonal hematopoietic disorders are useful tools to characterize diseases as well as to identify effective reagents for such diseases. To our knowledge, there is no cell line derived from TN-MPN although there are several lines with JAK2 V617F or CALR mutation. We have encountered an aggressive ET case with TN phenotype. Three years after initial diagnosis, blastic transformation occurred in this patient. Both conventional chemotherapy and allogeneic transplantation failed to cure the patient. After relapse, various therapies including ruxolitinib (JAK inhibitor) failed to control the disease. We have successfully established a cell line from the leukemic cells of this patient. We have also characterized the cell line by whole genome sequencing (WGS), RNA sequencing (RNA-Seq), as well as in vitro assays. 【Material and Methods 】 We have established a novel cell line, TNET-2, from the leukemic cells. WGS and RNA-Seq were performed at Beijing Genome Institute (BGI) Japan. Proliferation assays were performed in the presence or absence of ruxolitinib, AC220 (FLT3 inhibitor), giltertinib (FLT3/AXL inhibitor), etoposide, cytarabine or hydroxycarbamide. 【Results 】 The patient's leukemic cells have been cultured in RPMI1640 culture media with 10% fetal bovine serum and antibiotics. The cells were exponentially growing more than one year and the cell line, TNET-2, has been established. WGS has found 116 missense mutations in 110 genes encoding proteins. Also, five nonsense mutations causing early termination codons were found. InDel mutations causing frame-shifts were found in 28 genes. In-frame deletions were found in seven genes and in-frame insertions were found in nine genes. No mutations were found in JAK2, MPL, or CALR gene. A nonsense mutation was found in ASXL1 gene. Multiple genes encoding proteins associated with myeloid proliferation/differentiation, including CBL, A-RAF, HOXB9 and BCL11B, were mutated. Genes involved in DNA repair, for example ERCC6, were also mutated. Genes involved in apoptosis including CASP7, TRAK1 and SOCS3 were mutated. RNA-Seq revealed high expression of IRAK1 mRNA as well as low expression of TET2. Cell proliferation assays showed TNET-2 cells were resistant to ruxolitinib and conventional chemotherapeutic reagents including etoposide, cytarabine and hydroxycarbamide. However, both AC220 and giltertinib induced cell death. 【Discussion 】 It has been reported that alternations of ASXL1 and/or TET2 genes are common in TN-MPNs. Consistent with the reports, a nonsense mutation of ASXL1 gene and low expression of TET2 were detected in TNET-2 cells. Besides these well-known genetic changes, we have found more than 150 mutated genes. Some of them seem "actionable" / "targetable" mutations. There are no optimal treatments targeting an altered signaling pathway for TN-MPNs. Our novel cell line is a useful tool to characterize this subtype of MPN and find novel treatment options for this rare type of disease. In summary, we have, for the first time, established a cell line derived from TN-MPN and found novel altered signaling pathways by WGS, RNA-Seq and in vitro assays. Disclosures No relevant conflicts of interest to declare.