Herein, a rhodium-catalyzed highly chemo-, regio-, and stereo-selective linear cross coupling reaction of terminal alkynes with readily available 2,3-allenols afforded the synthetically and biologically versatile 3-alkyn-2(E)-enones/enals. The reaction exhibits a perfect match of the reactivities for these two classes of compounds since the well-known self-reacting products of both reactants were not formed. The reaction enjoys a wide substrate scope under ambient conditions. Furthermore, the synthetic potentials have been demonstrated by utilizing this reaction as a pivotal step: a very concise two-step total synthesis of naturally occurring isotaxifolial D has been achieved; an efficient synthesis of dehydroacitretin by applying this reaction two times has also been executed. Through mechanistic studies including D-labeling experiments, it is proposed that the reaction proceeded via terminal alkynylmetalation, stereodefined insertion providing a 2-alkynyl pi-allylic intermediate, which would undergo C-C single bond rotation, intramolecular ligand exchange, beta-H elimination, and reductive elimination resulting in 1,4-H delivery to afford the final enynes. The reaction circumvents the use of a stoichiometric amount of base under the catalysis of a single Rh catalyst since terminal alkynylmetalation is base free, and the catalyst is regenerated by aerobic oxidation in the presence of proton.

ABSTRACT:G protein-coupled receptor, class C, group 5, member D (GPRC5D) has emerged as a novel target for chimeric antigen receptor (CAR) T-cell therapy, demonstrating promising efficacy in multiple myeloma (MM). However, disease relapse is still common, and the mechanism of resistance remains poorly understood. In this study, we conducted whole-genome sequencing and whole-genome bisulfite sequencing on MM samples from 10 patients who relapsed after GPRC5D CAR T-cell therapy. Among these patients, 8 had GPRC5D loss, whereas 2 presented mixed expression (GPRC5D+/-). Genetic alterations were identified in 3 cases: one had a homozygous deletion in the GPRC5D gene, another had a biallelic loss in the regulatory regions of GPRC5D, and the third had homozygous deletions in both TNFRSF17 and GPRC5D after sequential anti-B-cell maturation antigen and anti-GPRC5D CAR T-cell therapies. No genetic changes were detected at GPRC5D locus in the remaining 7 cases. However, multiple hypermethylation sites were present in the transcriptional regulatory elements of the GPRC5D gene in 5 post-treatment MM samples. In MM cell lines, GPRC5D expression was inversely correlated with methylation levels in its regulatory regions. Furthermore, azacitidine treatment induced GPRC5D messenger RNA and protein expression in hypermethylated MM cell lines. Our findings highlight that biallelic genetic inactivation and hypermethylation-driven epigenetic silencing are key mechanisms contributing to GPRC5D loss and treatment resistance.

Photoinduced cyclization of 2,3-allenoic acids with sulfonyl chloride providing an efficient synthesis of 4-sulfonylated furan-2(5H)-ones under mild reaction conditions has been achieved. The reaction enjoys a high chemoselectivity and tolerates a wide range of functional groups. The catalytic cycle has been validated through control experiments, cyclic voltammetry studies, and Stern-Volmer quenching studies.

Here we report a conceptual protocol to construct 4-alkynyl furan-2(5H)-one derivatives efficiently through Rh(iii)/Cu(i) co-catalyzed highly regioselective oxy-alkynylation of 2,3-allenoic acids with terminal alkynes under mild aerobic conditions with atom economy. A wide variety of functional groups including biologically active groups have been tolerated. The synthetic potentials have also been demonstrated and the total syntheses of natural product appenolide A and its (Z)-isomer have been achieved by applying this protocol as the key step efficiently. A mechanism has been proposed based on mechanistic studies. An aerobic double functionalization of 2,3-allenoic acids with terminal alkynes has been achieved affording versatile 4-alkynylfuran-2(5H)-one derivatives, which have been demonstrated as platform molecules for synthesis of useful natural products.

Objective:To investigate the effects of down-regulation of p21 activated kinase 1(PAK1)on the proliferation,differentiation,and apoptosis of myeloproliferative neoplasm(MPN)cells(6133/MPL)with thrombopoietin receptor MPL mutation at codon 515(MPLW515L)and survival of 6133/MPL mice.Methods:Interference with the protein level of PAK1 in 6133/MPL cells was assessed using lentivirus-mediated shRNA transfection technology.CCK-8 assay was used to detect the effect of down-regulation of PAK1 on the proliferation viability of 6133/MPL cells,and colony-forming ability was measured by cell counting.Flow cytometry was used to detect the PAK1 kinase activity on the ability of polyploid DNA formation and cell apoptosis in 6133/MPL cells.The expression of cyclin D1,cyclin D3 and apoptosis-related protein Bax was detected by Western blot.The infiltration of tumor cells in spleen and bone marrow of 6133/MPL mice were detected by HE staining.Results:Down-regulation of PAK1 inhibited the proliferation and reduced the ability of cell colony formation of 6133/MPL cells.After knocking down PAK1,the content of polyploid DNA in 6133/MPL cells increased from 31.8 to 57.5％and 48.0％,and the proportion of apoptosis increased approximately to 10.8％.Down-regulation of PAK1 led to a reduction of infiltration of tumor cells in liver and bone marrow of 6133/MPL mice,thereby prolonging survival time.Conclusion:Down-regulation of PAK1 can significantly inhibit the growth of 6133/MPL cells,promote the formation of polyploid DNA,induce 6133/MPL cell apoptosis,and prolong the survival time of 6133/MPL mice.

A copper-catalyzed highly regioselective remote boryl- or silyl-cupration of allenynes, providing an efficient protocol for the synthesis of conjugated allene-substituted alkenyl boronates or silanes, has been developed.

The MPLW515L mutation is a prevalent genetic mutation in patients with myeloproliferative neoplasms (MPN), and utilizing this mutation in mice model can provide important insights into the disease. However, the relationship between intestinal homeostasis and MPN mice model remains elusive. In this study, we utilized a retroviral vector to transfect hematopoietic stem cells with the MPLW515L mutation, creating mutated MPN mice model to investigate their intestinal status. Our results revealed that the MPLW515L in MPN mice model aggravated inflammation in the intestines, decreased the levels of tight junction proteins and receptors for bacteria metabolites. Additionally, there was increased activation of the caspase1/IL-1β signaling pathway and a significant reduction in phos-p38 levels in the intestinal tissue in MPN mice. The MPLW515L mutation also led to up-expression of anti-microbial genes in the intestinal tract. Though the mutation had no impact on the alpha diversity and dominant bacterial taxa, it did influence the rare bacterial taxa/sub-communities and consequently impacted intestinal homeostasis. Our findings demonstrate the significance of MPLW515L mice model for studying MPN disease and highlight the mutation's influence on intestinal homeostasis, including inflammation, activation of the IL-1β signaling pathway, and the composition of gut microbial communities.

Most thrombopoietin receptor (MPL) mutations result in abnormal megakaryocyte expansion in the spleen or bone marrow (BM), leading to progressive fibrosis. It has been reported that p21 (Rac Family Small GTPase 1 [RAC1])-activated kinase 1 (PAK1) participates in the proliferation and differentiation of megakaryoblasts. PAK1 phosphorylation increased in patients with myeloproliferative neoplasms (MPNs) and murine MPN cells with the Mplw515l mutant gene in this study; however, the function of overactivated PAK1 in MPN cells remains unclear. We found that inhibition of PAK1 caused significant changes in the biological behaviors of MPLW515L mutant cells in vitro, including arrested growth or reduced clonality and increased polyploid DNA and cell apoptosis due to upregulated cleaved caspase 3. In vivo, PAK1 inhibitor treatment caused a slow elevation of leukocytosis and hematocrit (HCT) and a reduction in hepatosplenomegaly in 6133/MPLW515Ltransplanted mice, along with reduced tumor cell infiltration and prolonged survival. Further, deletion of PAK1 sustained a relatively normal HCT and platelet count at the beginning of the disease but did not completely alleviate the splenomegaly of MPLW515L mutant mice. Notably, PAK1 knockout attenuated the destruction of splenic structure, and reduced the megakaryocyte burden within the BM. These results suggest that inhibition of PAK1 may be a useful method for treating MPLW515L mutant MPN by intervening megakaryocytes. (c) 2023 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

OBJECTIVE:To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation, and establish a systematic evaluation system to verify the success of model construction.METHODS:The bone marrow c-kit+ cells of the mice were obtained by the following steps: The mice were killed by cervical dislocation, the femur, tibia and ilium were separated, and the bone marrow cells were collected. The c-kit+ cells were sorted after incubation with CD117 magnetic beads. The method of constructing mouse primary mutant cells is as follows: A gene mutation vector with a GFP tag was constructed by the retroviral system, and the retroviral vector was packaged into the Platinum-E cells to obtain the virus supernatant, and then used it to infect the c-kit+ cells of mice. The MPN mouse model was constructed as follows: the mouse primary c-kit+ cells containing the mutant genes were collected after infection, and then transplanted them via the tail vein into the female recipient mice of the same species which were irradiated with a lethal dose of gamma rays (8.0 Gy). The MPN mouse model was evaluated as follows: After transplantation, the peripheral blood of the mice was regularly collected from the tail vein to perform the complete blood count test, and the size of spleen and the degree of bone marrow fibrosis were estimated.RESULTS:The mouse c-kit+ cells with the mutant genes were successfully obtained from the bone marrow. MPN mouse model was successfully constructed: The peripheral blood cells of the MPN-transplanted mice carried exogenous implanted GFP-positive cells, and the white blood cells (WBC), platelet (PLT) and hematocrit (HCT) were all increased; the body weight loss, and the water and food intake were reduced in the transplanted mice; further pathological analysis showed that the transplanted mice displayed splenomegaly and bone marrow fibrosis. These results suggested that the MPN mouse model was successfully constructed. According to the common and different characteristics of the three MPN mouse model, a preliminary evaluation system for judging the success of MPN mouse model construction was summarized, which mainly included the following indicators, for example, the proportion of GFP-positive cells in the peripheral blood of mice; WBC, PLT and HCT; the degree of spleen enlargement and the bone marrow fibrosis.CONCLUSION:The MPN mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation is successfully established by retroviral system, which can provide an important experimental animal model for the research of MPN pathogenesis and drug-targeted therapy.

<p>Supplementary figures and figure legends</p>

Comprehensive Summary Aerobic oxidation has been catching more and more attention because of its atom economy and environmental friendliness. Oxidation of diols is a challenge due to various oxidative products. Thus, highly selective aerobic oxidation affording specific products is of current interest. In this work, a combination of Fe(NO 3 ) 3 · 9H 2 O/TEMPO/KCl catalysis has been identified as an efficient recipe for the aerobic oxidation of 1,4‐diols affording γ‐butyrolactones under mild conditions. The reaction exhibits decent chemo‐ and regioselectivity of symmetrical and unsymmetrical 1,4‐diols. The optically active γ‐lactones may also be prepared from optically active 1,4‐diols without erosion of the ee via this method. Furthermore, this approach was successfully applied to synthesize NBP, a commercial drug.

AbstractPurpose: The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. Experimental Design: We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage–committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. Results: JAK2V617F-mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. Conclusions: Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

Objective:To investigate the effects of down-regulation of aurora kinase (AURK) B on the proliferation and apoptosis of CALR-mutated MARIMO cells, as well as changes in related genes and signalling. Methods:MARIMO cells derived from a 68-year-old female patient with CALR mutation-positive myeloproliferative neoplasms(MPN) were selected for the study. Lentivirus-mediated short hairpin RNA (shRNA) was used to transfect MARIMO cells, and the cells were categorized into sh-AURKA group, sh-AURKB group and control shRNA (sh-CTRL) group according to the difference of shRNA. The MARIMO cell lines in each group were observed by inverted fluorescence microscope, and their green fluorescent protein (GFP) positivity was detected by flow cytometry (FCM). The reproduction and apoptosis of MARIMO cells in sh-AURKB group and sh-CTRL group were analyzed by FCM, 5-ethynyl-2′-deoxyuridine (EdU)/4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and Annexin V/PE reagent. The enrichment of differentially expressed genes (DEG) in sh-AURKB group and sh-CTRL group was analyzed by RNA sequencing (RNA-Seq), and gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to screen for significant DEG. The relative expression of mRNA and proteins of DEG were validated by real-time fluorescence quantitative-PCR (RT-qPCR) and Western blotting. Relative expression levels were verified. The comparison of measurement data between two groups was performed by t test. The overall comparison among 3 groups were conducted by one-way analysis of variance, pairwise comparisons between groups were conducted by least-significant difference (LSD) test. This study was in line with World Medical Association Declaration of Helsinki revised in 2013. Results:① The number of MARIMO cells 72 h after transfection was reduced in sh-AURKB group compared with sh-CTRL group [ (0.62±0.03) ×10 5vs (12.44±0.08) ×10 5], and the difference was statistically significant ( t=257.20, P<0.000 1).Compared with sh-CTRL group, the proportion of MARIMO cells in the S-stage in sh-AURKB group was decreased [(33.37 ± 0.75)% vs (42.77 ± 0.91)%], and the proportion of MARIMO cells in G2-M stage was increased [(27.83 ± 0.69)% vs (17.90 ± 0.40)%], the proportion of apoptosis was increased [(16.47 ± 0.70)% vs (2.75 ± 0.13)%], all the differences were statistically significant ( t=13.83, P=0.000 2; t= 23.78, P<0.000 1); t=33.28, P<0.000 1). ② RNA-Seq assay showed that compared to sh-CTRL group, there were 2 787 genes up-regulated and 2 420 genes down-regulated in MARIMO cells in sh-AURKB group. ③ The DEG of sh-AURKB group and sh-CTRL group were mainly annotated in GO nodes such as intracellular region, intracellular membrane border organelle, membrane border organelle, intracellular organelle part, organelle part, etc.. KEGG enrichment analysis showed that a total of 89 signal pathways were significantly enriched. Further screening of DEG-related pathways showed that the 2 groups of significant DEG were NDUFV1, NPRL2, MAP2K4, and CPEB1. ④ RT-qPCR results showed that the relative mRNA expression levels of NPRL2, MAP2K4 and CPEB1 in sh-AURKB group were lower than those in sh-CTRL group, and the differences were statistically significant (LSD- t=14.13, 9.13, 3.10; P<0.000 1, <0.000 1, =0.021). Western blotting results showed that compared with SH-CTRL group, the protein strip grayscale value of NDUFV1/GAPDH in sh-AURKB group increased, while NPRL2/GAPDH, CPEB1/GAPDH, MAP2K4/GAPDH and p-MAP2K4/GAPDH reduced, and the differences were statistically significant (LSD- t=22.60、12.09、7.48、20.48、8.22, P<0.000 1、<0.000 1、=0.000 3、<0.000 1、=0.000 2) Conclusions:AURKB is involved in the proliferation, differentiation and apoptosis of CALR mutant MARIMO cells, and down-regulation of AURKB could induce significant changes in gene expression levels. DEG are mainly enriched in the cell metabolism, cell cycle and apoptosis related pathways.

A [Cp*RhCl 2 ] 2 catalyzed three-component coupling cyclization of 2,3-allenols and 2,3-allenoic acids affording furanone derivatives with an acetate functionality was established. Isotopic labelling experiments unveiled a unique mechanism.

Since the important role of inflammation in myeloproliferative neoplasms (MPN) progression, inflammasome is an important regulator of inflammation involved in the development of tumor. Here, we aimed to investigate the function of NLRP6 in bone marrow (BM) microenvironment in MPN in the hope of getting better treatment for MPN. We first analyzed that NLRP6 was highly expressed in BM mononuclear cells from individuals newly diagnosed with ET, PV, PMF patients (with or without JAK2 V617Fmutation). Similar results were obtained in BM cells and BM stromal cells of MPL W515L transduced C57BL/6 mice (C57BL/6 MPN). The results were validated that NLRP6 of the BM microenvironment was involved in the development of MPN. We thus hypothesized that the absence of NLRP6 in BM microenvironment affects the development of MPN. NLRP6 -/- mice and C57BL/6 mice MPN models were established by the same method. The experiments demonstrated that overall survival of NLRP6 -/- MPN mice was significantly prolonged compared to C57BL/6 MPN mice. Loss of NLRP6 in BM microenvironment interfered with hematopoiesis of BM, but it had no effect on the extramedullary hematopoiesis (EMH) in spleen. Pathological analysis showed that the destruction of spleen structure in NLRP6 -/- MPN mice and the infiltration of atypical megakaryocytes were less than C57BL/6 MPN mice. The tumor load in spleen was significantly reduced and the structure of spleen was intact, the hematopoietic function of the BM returned to normal and the tumor load in the BM was significantly reduced at 5 weeks in NLRP6 -/- MPN mice after sequential transplantation. It suggested that knockout of NLRP6 in the BM microenvironment significantly attenuated the tumorigenicity of malignant HSCs, delayed or even inhibited the development of MPN disease. The expression of inflammatory cytokines, abnormal megakaryocytes and the phenomenon of aggregation and reticular fibers in BM microenvironment were significantly reduced in NLRP6 -/- MPN group compared with the control group. After sequential transplantation, the destruction of BM structure was alleviated, megakaryocytes aggregation and heteromorphic megakaryocytes in BM were reduced, and reticular fibers were significantly diminished. Furthermore, the cytokines promoting disease progression in BM microenvironment were significantly reduced in NLRP6 -/- MPN group, suggesting that knockout of NLRP6 can improve the BM microenvironment and alleviate the progression of myelofibrosis. Finally, to gain better insight into the role of NLRP6 inflammasome in MPN pathogenesis, we sought to dissect the changes with scRNA-seq and pathway identification based on available data and bioinformatic pathway analysis in NLRP6 -/- mice. The results of scRNA-seq analysis showed that C18 population of malignant HSCs of recipients in primary transplantation was added, C16, C13 and C9 cell populations disappeared after deletion of NLRP6 in BM microenvironment, while C5, C6, C7, C10 and C12 cell populations increased significantly. Regulon of various subpopulations of malignant HSCs changed significantly, mainly involving cell development and differentiation, immune response, inflammatory BM microenvironment, vascular endothelial homeostasis and tumor related genes. Our results indicate that knockout of NLRP6 in the BM microenvironment may affect the progression of MPN by altering some malignant HSCs subpopulations and regulons, participating in immune, inflammatory, tumor metabolism and other signaling pathways to regulate the functional characteristics of tumor stem cells of MPN.Our data here provide new evidence for NLRP6 as potential target in the treatment of MPN.

Allenenitriles bearing different synthetically versatile functional groups have been prepared smoothly from 5-alkynyl fluorosulfonamides in decent yields with an excellent chemo- and regio-selectivity under redox neutral conditions. The resulting allenenitriles can be readily converted to useful functionalized heterocycles. Based on mechanistic study, it is confirmed that this is the first example of radical-based non-activated propargylic C-H functionalization for allene syntheses.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Butafulvene is a constitutional isomer of benzene, comprising a cyclobutene skeleton bearing two exocyclic conjugated methylene units. As a result of the intrinsic high strain energy and anti-aromaticity, the preparation of butafulvene compounds has been a fundamental issue for the development of butafulvene chemistry. Here an efficient palladium-catalysed coupling protocol involving propargylic compounds has been developed, providing a solid and versatile strategy for the rapid assembly of symmetric butafulvene derivatives. Based on mechanistic studies, two complementary mechanisms, both involving palladium catalysis, have been confirmed. With the mechanism unveiled, the synthesis of non-symmetric butafulvenes has also been achieved. Advantages of this strategy include tolerance to a wide range of propargylic molecules, mild reaction conditions, simple catalytic systems and easy scalability. The synthetic potential of the products as platform molecules for cyclobutene derivatives has also been demonstrated.

A copper-catalyzed three-component reaction of cyclobutanone oxime esters and 1,3-enynes in the presence of TMSCN or TMSCF3 has been developed. This mild protocol enjoys a broad substrate scope tolerating many functional groups, providing a facile access to 1,7-double-functionalized allenes, which are difficult to prepare. The allenyl nitrile products may be easily transformed into allenoic acid derivatives and stereodefined tetrasubstituted alkenes, demonstrating their potentials as platform molecules in synthesis. A mechanism has been proposed on the basis of mechanistic studies.

Herein we report a [Cp*RhCl2]2-catalyzed coupling cyclization of two different classes of allenes with 2,3-allenoic acids affording 2(5H)-furanone skeletons of products and 2,3-allenols forming a conjugated (E)-enal or enone functionality to the β-position of the 2(5H)-furanones. These products are important building blocks for the syntheses of potentially bioactive compounds. The reaction proceeded via the nucleometalation, insertion, and stereodefined 1,4-H delivery carried by rhodium.