Murine Leukemia Cells Research Articles

Combined Genetic Lesions in TP53 and CDKN2A/CDKN2B Drive B Cell Receptor-Dependent/Costimulatory Signal-Independent Proliferation in Richter Syndrome

B cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitro upon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptor (TLR) ligands or T cells. However, the rapid reduction in the percentage of proliferating CLL cells in patients treated with a BTK or SYK inhibitor suggests that the BCR may be more directly involved, at least in a subset of cases. To further address this issue, we investigated the expression of cell cycle regulatory proteins in human and Eμ-TCL1-derived murine CLL cells stimulated with immobilized anti-IgM or cognate antigen, respectively. In both cases, BCR stimulation induced the expression of the positive cell cycle regulators MYC, CCND1, CCND2 and CDK4, consistent with G1 cell cycle entry, but also induced the expression of the negative regulators CDKN1A, CDKN2A and CDKN2B, which block cell cycle progression. Since combined deficiency of these negative regulators occurs in approximately one quarter of Richter syndrome cases because of deletion of the CDKN2A/CDKN2B locus and inactivating genetic lesions of the CDKN1A regulator TP53, we introduced these defects by CRISPR/Cas9 in autoreactive murine Eμ-TCL1 leukemia cells and investigated the effects on leukemia behavior. Adoptive transfer experiments showed that combined targeting of TP53, CDKN2A and CDKN2B results in accelerated leukemia growth and morphological changes consistent with Richter's transformation, including more diffuse infiltration, larger and more pleomorphic cells, and a higher proliferation rate. Moreover, in vitro experiments showed that cells with combined TP53/CDKN2A/2B deficiency had acquired the capacity for spontaneous proliferation, in contrast to control, TP53- or CDKN2A/2B-targeted cells which died after a couple of weeks in culture. Nucleotide sequencing of the TP53/CDKN2A/2B-targeted cells showed complete disappearance of the wild type alleles, suggesting that biallelic loss of all three genes is required for spontaneous growth in vitro. Combined disruption of TP53, CDKN2A and CDKN2B in two other autoreactive Eμ-TCL1 leukemias yielded the same results. To determine whether the spontaneous in vitro proliferation is dependent on BCR signals, the TP53/CDKN2A/2B-deficient cells were transfected with Cas9 ribonucleoprotein complexes targeting the IgM heavy chain constant region (IGHM) gene or were treated with the BCR inhibitors ibrutinib, idelalisib and fostamatinib. Disruption of the IGHM gene or treatment with a BCR inhibitor resulted in almost complete block of proliferation. In contrast, knockdown of the TLR-adaptor protein MyD88 had no effect. Considering that T cells were not present in the culture conditions, these experiments establish that proliferation of Eμ-TCL1 leukemia cells with biallelic TP53/CDKN2A/2B disruption is BCR-dependent but independent of costimulatory signals. To validate these findings in a human setting, we performed experiments with two recently established Richter syndrome patient-derived xenografts, one with biallelic inactivation/deletion of TP53, CDKN2A and CDKN2B (RS9737), and one with wild type TP53, CDKN2A and CDKN2B (RS1316). BrdU incorporation experiments showed that only RS9737 cells proliferate in vitro following BCR stimulation, whereas both RS9737 and RS1316 cells proliferate following stimulation with CpG + IL-15 or CD40L + IL-4 + IL-21. Finally, we tested the activity of combined treatment with a BCR inhibitor and the CDK4/6 inhibitor palbociclib against the murine and human TP53/CDKN2A/2B-deficient Richter syndrome models. Combined treatment showed synergistic activity in vitro and significantly prolonged mouse survival in vivo compared to single agent treatment (n = 10 mice/group, P<0.001). In conclusion, these data provide evidence that BCR signals are directly involved in regulating CLL cell proliferation and suggest that frequently co-occurring genetic lesions in TP53 and CDKN2A/2B contribute to Richter transformation by allowing for BCR dependent/costimulatory signal independent proliferation, which can be therapeutically targeted with a BCR and CDK4/6 inhibitor combination. Disclosures Deaglio: Verastem: Research Funding; Heidelberg Pharma: Research Funding. Laurenti:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees. Efremov:Janssen-Cilag International: Speakers Bureau.

Transcriptional Control of HP1a By the RNA Binding Proteins Rbmx/L1 Maintain Chromatin State in Myeloid Leukemia

Mutations and aberrant expression of RNA binding proteins (RBPs) have recently been found to contribute to leukemia development (Prieto and Kharas, CSH, 2020). Previously we have identified the RBP RBMX (RNA binding motif protein, X-linked) in our shRNA in vivo screen using murine MLL-AF9 driven leukemia (Prieto et al, ASH abstract 2018). Here we uncover the role of RBMX and its retrogene RBMXL1 in normal hematopoiesis and leukemogenesis in mouse and human systems. To investigate RBMX function in normal hematopoiesis, we created an Rbmx Mx1-Cre conditional knockout model to specifically delete Rbmx in the hematopoietic system. We gender stratified our studies since Rbmx is sex-linked. Deletion of Rbmx in both female and male primary mice as well as in non-competitive transplant animals did not affect hematopoiesis. Additionally, Rbmx knockout (KO) leads to a mild reduction in multipotent progenitors (MPP2 and MPP4) in female competitive transplanted mice although no defects in long-term hematopoiesis was observed in male competitive transplanted mice. These data suggest that Rbmx is dispensable for normal hematopoiesis. To identify the role of RBMX in leukemogenesis we knockout Rbmx in MLL-AF9 murine leukemia cells and found it significantly reduced colony formation in vitro and delayed leukemogenesis in vivo, indicating that Rbmx is required for leukemia maintenance. We observed, however, that MLL-AF9 transformed cells from Rbmx KO donor mice showed no delay in leukemia initiation versus cells from wildtype. We then determined that while Rbmx deletion is effective with complete depletion of mRNA, the retrogene RBMXL1 expression is maintained in Rbmx deficient cells, which may compensate for Rbmx deletion in leukemia initiation. Indeed, depletion of RBMXL1 by shRNAs (KD) in Rbmx deficient leukemia cells results in a drastic reduction in colony formation, increased colony myeloid differentiation, and induced apoptosis in cells deficient for both RBMXL1 and RBMX compared to those only depleted of RBMX. Correspondingly, RBMXL1 KD in Rbmx deficient leukemia cells resulted in further delayed leukemogenesis in vivo, indicating that RBMXL1 is functionally redundant to RBMX and both genes are required for leukemia development and maintenance. We next investigated the role of RBMX/L1 in normal human hematopoietic and leukemia cells. We found that RBMX/L1 expression were higher in AML cell lines (n=10/11) and primary AML patient samples (n=2/4) compared to healthy individuals. RBMX/L1 KD by shRNAs led to a dramatic decrease in cell proliferation, induction of apoptosis and myeloid differentiation in several human myeloid leukemia cell lines (MOLM-13, THP-1, K562, and KCL-22). Additionally, RBMX/L1 depletion significantly delayed leukemogenesis in vivo of AML cell lines (median survival of 51.5 days in control vs. median 'not reached' in shRNA1 and shRNA2), and of primary leukemia cells derived from an AML patient (median 50 days in control vs. "not reached" in shRNA2). We next depleted RBMX/L1 in human CD34+ stem and progenitor cells and observed reduced colony formation but no increase in apoptosis. Taken together, our data suggest that RBMX/L1 are differentially required in leukemia cells versus normal cells in both human and mouse systems. To uncover the mechanism of RBMX/L1 function, we performed complex chromosomal karyotyping analysis of RBMX/L1 depleted MOLM13 cells and revealed increased metaphases with breaks and gaps. In addition, ATAC-seq analysis showed profound genome-wide changes in chromatin accessibility and compaction upon RBMX/L1 depletion. Analyses of RNA-seq and transcriptome-wide RBMX/L1 binding targets (PAR-CLIP; Liu et al. 2017) revealed that RBMX/L1 directly binds to mRNAs and affects transcription of multiple loci including the heterochromatin protein 1 alpha (HP1a) mRNA. Using single molecule RNA FISH, we uncovered that RBMX/L1 controls the nascent transcription of the HP1a locus. Forced HP1a expression rescued the RBMX/L1 depletion effects on cell growth and apoptosis. Overall, we determine that RBMX/L1 control leukemia cell survival by regulating chromatin state through its downstream target HP1a. This study describes a novel genetic compensation phenomenon in leukemia and illuminate a mechanism for RBPs directly promoting transcription. Our results suggest RBMX/L1 as well as HP1a as potential novel therapeutic targets in myeloid malignancies. Disclosures Steidl: Aileron Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Stelexis Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Research Funding; Pieris Pharmaceuticals: Consultancy. Kharas:Accent Therapeutics: Consultancy; 28-7: Research Funding.

Genomic Analysis of Congenital Myeloid Sarcoma Identifies Significant Bone Marrow Involvement in the Absence of Morphologic Blasts

Myeloid sarcoma is a tumor mass consisting of blasts at extramedullary sites, most frequently the skin and soft tissues. Skin involvement, also termed leukemia cutis, is a well-described entity in congenital leukemia and is present in approximately 50% of patients diagnosed within the first 4 weeks of life. Most cases of congenital leukemia cutis harbor KMT2A rearrangements, as do up to 80% of cases of infantile leukemia, and are typically accompanied by bone marrow involvement. Here we describe a rare CIC-rearrangement as the driver mutation for a case of congenital cutaneous myeloid sarcoma in a newborn female who was noted at birth to have a violaceous, nodular rash. Skin biopsy of the nodular rash shortly after birth with immunohistochemical staining was consistent with myeloid sarcoma. Bilateral bone marrow aspirates and biopsies were also performed, but failed to reveal an aberrant blast population by morphology and flow cytometry. Cytogenetics of the cutaneous myeloid sarcoma was significant for t(10;19)(q23;q13.2) and fluorescence in situ hybridization performed on the tumor confirmed a CIC-rearrangement. CIC-rearranged sarcomas are a new entity of undifferentiated small round cell sarcoma characterized by fusion events involving the CIC gene but are not a known to be driver mutations in myeloid neoplasms. Despite the absence of a blast population in the bone marrow, 75% of analyzed metaphases carried the t(10;19)(q23;q13.2). To further explore the underlying genomic events, whole genome, exome, and transcriptome sequencing was performed on both sarcoma and bone marrow specimens. Sequencing revealed an in-frame CIC-NUTM2A fusion gene present in both the skin and the bone marrow which has been previously described in a single case of undifferentiated soft tissue sarcoma. There were no large-scale chromosomal losses or gains and no copy neutral loss of heterozygosity events. Two exonic single nucleotide variations (SNVs) were detected, both of which were limited to the skin sarcoma and not predicted to be pathogenic. In vitro and in vivo modeling demonstrated that the CIC-NUTM2A fusion protein was highly leukemogenic. Colony forming assays performed using transduced murine bone marrow revealed that the CIC-NUTM2A fusion conferred self-renewal in contrast to CIC, NUTM2A, and the reciprocal NUTM2A-CIC product which failed to serially replate (p<0.001). Transplantation of CIC-NUTM2A modified murine bone marrow cells resulted in a fully penetrant myeloid leukemia with a median survival of 21 days in primary transplants and 16 days in secondary transplants. Transcriptome analysis of these tumors revealed a distinct gene expression profile when compared to several classic myeloid associated fusion genes including AML1-ETO and MLL-AF6. To further understand the acquisition of a malignant phenotype by morphology between the bone marrow and the sarcoma, targeted deep sequencing was performed for all tier 1, 2 and 3 mutations identified by whole genome and exome sequencing. A shared ancestral clone was identified in both specimens along with three subclones specific to the sarcoma. Four SNVs acquired in the sarcoma were found to be present in regulatory regions of genes that were also differentially expressed between the sarcoma and the bone marrow including C6orf120, SMURF1, TJAP1, and PID1. PID1 was found to be downregulated in the malignant sarcoma specimen and has been previously shown to be a regulator of the AKT/PI3K pathway. Low PID1 expression has been associated with poor outcomes in other malignancies, including pediatric glioblastoma. A genome wide CRISPR screen of our CIC-NUTM2A positive murine leukemia cells revealed an enrichment for PID1 deficient cells as well as PTEN confirming the cooperativity between CIC-NUTM2A and the AKT/PI3K pathway (p=0.009 and p=0.005 respectively). In conclusion, we describe a rare fusion gene, CIC-NUTM2A, which leads to an aggressive myeloid malignancy in both humans and mice. Targeted deep sequencing demonstrated the clonal evolution from the bone marrow and acquisition of a cooperating mutation targeting the AKT/PI3K pathway in a subset of extramedullary cells that led to morphologic transformation. A thorough interrogation of the bone marrow in patients with myeloid sarcoma is warranted even in the absence of morphologic and immunophenotypic blasts by flow cytometry to optimally track residual disease during treatment. Disclosures No relevant conflicts of interest to declare.

Antioxidant Effects and in vitro Cytotoxicity on Human Cancer Cell Lines of Flavonoid-Rich Flamboyant (Delonix regia (Bojer) Raf.) Flower Extract.

Cancer is a non-communicable disease with increasing incidence and mortality rates worldwide, including Thailand. Its apparent lack of effective treatments is posing challenging public health issues. Encouraging research results indicating probable anti-cancer properties of the Delonix regia flower Extract (DRE) have prompted us to evaluate the feasibility of developing a type of product for future cancer prevention or treatment. In the present report, using High-Performance Liquid Chromatography (HPLC), we demonstrate in the DRE the presence of high concentrations of three identifiable flavonoids, namely rutin 4.15±0.30% w/w, isoquercitrin 3.04±0.02 %w/w, and myricetin 2.61±0.01% w/w, respectively, while the IC50 of DPPH and ABTS assay antioxidation activity was 66.88±6.30 μg/ml and 53.65±7.24 μg/ml, respectively. Our cancer cell line studies using the MTT assay demonstrated DREs potent and dosedependent inhibition of murine leukemia cell line (P-388: 35.28±4.07% of cell viability remaining), as well as of human breast adenocarcinoma (MCF-7), human cervical carcinoma (HeLa), human oral cavity carcinoma (KB), and human colon carcinoma (HT-29) cell lines in that order of magnitude. Three identifiable flavonoids (rutin, isoquercitrin and myricetin) with high antioxidation activity and potent and dose-dependent inhibition of murine leukemia cell line and five other cancer cell lines were documented in the DRE. The extract's lack of cytotoxicity in 3 normal cell lines is a rare advantage not usually seen in current antineoplastic agents. Yet another challenge of the DRE was its low dissolution rate and long-term storage stability, issues to be resolved before a future product can be formulated.

Clinical MDR1 inhibitors enhance Smac-mimetic bioavailability to kill murine LSCs and improve survival in AML models

AbstractThe specific targeting of inhibitor of apoptosis (IAP) proteins by Smac-mimetic (SM) drugs, such as birinapant, has been tested in clinical trials of acute myeloid leukemia (AML) and certain solid cancers. Despite their promising safety profile, SMs have had variable and limited success. Using a library of more than 5700 bioactive compounds, we screened for approaches that could sensitize AML cells to birinapant and identified multidrug resistance protein 1 inhibitors (MDR1i) as a class of clinically approved drugs that can enhance the efficacy of SM therapy. Genetic or pharmacological inhibition of MDR1 increased intracellular levels of birinapant and sensitized AML cells from leukemia murine models, human leukemia cell lines, and primary AML samples to killing by birinapant. The combination of clinical MDR1 and IAP inhibitors was well tolerated in vivo and more effective against leukemic cells, compared with normal hematopoietic progenitors. Importantly, birinapant combined with third-generation MDR1i effectively killed murine leukemic stem cells (LSCs) and prolonged survival of AML-burdened mice, suggesting a therapeutic opportunity for AML. This study identified a drug combination strategy that, by efficiently killing LSCs, may have the potential to improve outcomes in patients with AML.

In vitro evaluation of immunobiological activity of simple mannolipids

Immunomodulation, cytotoxicity and anti-cancer activity of selected amphiphilic non-ionic (thio)alkyl α-D-mannosides (with aglycone of C6-C12) were investigated in vitro in human cervix epitheloid carcinoma cell line HeLa, murine melanoma cancer cells B16, murine lymphocytic leukemia cell line L1210, murine fibroblast cell line NIH 3 T3 and murine macrophage cell line RAW 264.7. Toxicological studies revealed structure-dependent immunobiological effectivity based on a tight interaction with relevant cells. The results demonstrated diverse immunomodulation of macrophage cell-line RAW264.7 proliferation and production of Th1 and Th2 cytokines, and induction of pro-inflammatory interleukins IL-1α, TNFα, IL-6, IL-12 and IL-17 and anti-inflammatory IL-10 following (thio)alkyl α-D-mannosides 24 and 48 h exposure. Direct application of alkyl mannosides MOC10 and MOC12 and their thio analogues MSC10 and MSC12 in reconstructed human EpiDerm™ and MOC12 and MSC12 in EpiOcular™ model assays for dermal and ocular irritation together with quantification of human proinflammatory cytokines IL-1α, TNFα, IL-6 and IL-8 culture media release was used to ascertain toxicological safety.

Study of biomedical properties of Rhizophora mucronata fruit from Rembang, Central Java

Indonesian in coastal region utilize mangrove ecosystem for aquaculture, fishing ground, firewood source, tourism, nevertheless it is rarely reported as a source of biomedical substances. Rhizophora mucronata is one of the most common mangrove species in Rembang, Central Java. However, study of biomedical prospect of its fruit is neglected. This study aimed to investigate the antimicrobial activity of R. mucronata fruit extract to inhibit human pathogens (E. coli, S. aureus, M. luteus, T. rubrum, and C. albicans), as well as its anticancer activity against P388 murine leukemia cells. Metabolite profile was characterized through phytochemical test and HPLC. The result showed there was no antimicrobial activity of R. mucronata fruit extract against all human pathogens. Cytotoxic assay indicated a moderate anticancer potential with IC50 value of 398 μg/mL. Phytochemical test indicated the presence of alkaloid, flavonoid, and steroid/triterpenoid in the crude extract.

Identification of marine bacteria HPP.4A and HPP.T13 and its anticancer activity against P388 murine leukaemia cell

Sea cucumber has been widely studied as a source of bioactive compounds with various biological activities such as antibacterial, antifungal, antioxidant, and anticancer. However, there are a few studies have reported on the biological activity of its associated bacteria. The purpose of this study were to determine the potential of sea cucumber associated bacteria from Panjang Island, Jepara, Central Java, Indonesia as a natural source of anticancer compounds and identify the prospective isolates through DNA barcoding. Bacteria HPP.4A and HPP.T13 were isolated from the gut of sea cucumber Holoturia atra. The bacteria were cultivated in three different media (A3, A11, and A16) then extracted using 1-butanol with maceration method. Cytotoxic assay of each extract was conducted against P388 murine leukaemia cell. Bacteria HPP.4A and HPP.T13 were identified through molecular approach as Sallinicoccus roseus and Sphingobium yanoikuyae with 99.73% similarity. The strongest anticancer activity was showed by Sallinicoccus roseus extract which cultivated in A11 medium while Sphingobium yanoikuyae extract in A3 medium.

Construction and efficacy evaluation of novel swine leukocyte antigen (SLA) class I and class II allele-specific poly-T cell epitope vaccines against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease. Here we report the development of subunit PRRSV-2 vaccines by expressing swine leucocyte antigen (SLA) class I and class II allele-specific epitope antigens in a robust adenovirus vector. SLA I-specific CD8 and SLA II-specific CD4 T cell epitopes of PRRSV-2 NADC20 were predicted in silico. Stable murine leukaemia cell lines (RMA-S), which are TAP-deficient and lacking endogenous class I epitope loading, were established to express different SLA I alleles. The binding stability of PRRSV T cell epitope peptides with SLA I alleles expressed on RMA-S cells was characterized. Two PRRSV poly-T cell epitope peptides were designed. NADC20-PP1 included 39 class I epitopes, consisting of 8 top-ranked epitopes specific to each of 5 SLA I alleles, and fused to 5 class II epitopes specific to SLA II alleles. NADC20-PP2, a subset of PP1, included two top-ranked class I epitopes specific to each of the five SLA I alleles. Two vaccine candidates, Ad-NADC20-PP1 and Ad-NADC20-PP2, were constructed by expressing the polytope peptides in a replication-incompetent human adenovirus 5 vector. A vaccination and challenge study in 30 piglets showed that animals vaccinated with the vaccines had numerically lower gross and histopathology lung lesions, and numerically lower PRRSV RNA loads in lung and serum after challenge compared to the controls, although there was no statistical significance. The results suggested that the Ad-NADC20-PP1 and Ad-NADC20-PP2 vaccines provided little or no protection, further highlighting the tremendous challenges faced in developing an effective subunit PRRSV-2 vaccine.

Antibacterial, cytotoxicity and metabolite profiling of crude methanolic extract from andaliman (Zanthoxylum acanthopodium) fruit

Abstract. Sibero MT, Siswanto AP, Frederick EH, Wijaya AP, Syafitri E, Farabi K, Murwani R, Saito S, Igaras Y. 2020. Antibacterial, cytotoxicity and metabolite profiling of crude methanolic extract from andaliman (Zanthoxylum acanthopodium) fruit. Biodiversitas 21: 4147-4154. The local community in North Sumatra has utilized andaliman fruit (Zanthoxylum acanthopodium) as spices for traditional cuisines because it has a unique flavor. Information on the antimicrobial activity of Z. acanthopodium fruit against aquaculture pathogens and its bioactivity against leukemia cell lines are limited. The purposes of this study were to evaluate the antimicrobial activity of Z. acanthopodium fruit against Tenacibaculum maritimum, Vibrio alginolyticus, V. anguillarum, V. harveyi that are known as pathogens in aquaculture; to determine cytotoxic property against murine P388 leukemia cells; and to characterize its metabolites profile. The sample was extracted using methanol by the maceration method. Antibacterial assay was conducted by Kirby-Bauer disc diffusion method; while cytotoxicity assay using the XTT method. Proximate analysis showed that Z. acanthopodium fruit contained 63.41% of moisture, 24.73% of crude fiber, 9.81% of crude protein, 6.90% of ash, and 2.55% of crude fat. Several phytochemical components were detected, such as alkaloid, flavonoid, tannin, triterpenoid, and steroid. The GC/MS analysis indicated the presence of various compounds from terpenoid and terpenes derivatives. This study indicated that Z. acanthopodium fruit was not potential as antibacterial agents against the aquaculture pathogens; however, the methanol extract showed cytotoxic potential with IC50 19.14 µg/mL against murine P388 leukemia cells.

Compounds 7-Hydroxy-6-Metoxic Cytotoxic Cytotoxic Stem Leather From Chisochetonmacrophyllus (Meliaceae) Bark

A phenyl propanoid compound, 7-hydroxy-6-methoxycoumarin has been isolated from the bark of Chisocheton macrophyllus (Meliaceae). The chemical structure of isolated compound was identified on the basis of spectroscopic data including UV, IR, 1D-NMR, 2D-NMR and mass along with comparison with those spectral data previously reported. 7-hydroxy-6-methoxycoumarin compound showed cytotoxic effect against P-388 murine leukemia cell with IC50 value of 16.5 mg/mL. 7-hydroxy-6-methoxycoumarin compound was reported for the first time from genus of Chisocheton.

Compounds 7-Hydroxy-6-Metoxic Cytotoxic Cytotoxic Stem Leather From Chisochetonmacrophyllus (Meliaceae) Bark

A phenyl propanoid compound, 7-hydroxy-6-methoxycoumarin has been isolated from the bark of Chisocheton macrophyllus (Meliaceae). The chemical structure of isolated compound was identified on the basis of spectroscopic data including UV, IR, 1D-NMR, 2D-NMR and mass along with comparison with those spectral data previously reported. 7-hydroxy-6-methoxycoumarin compound showed cytotoxic effect against P-388 murine leukemia cell with IC50 value of 16.5 mg/mL. 7-hydroxy-6-methoxycoumarin compound was reported for the first time from genus of Chisocheton.

Identification, Purification and Molecular Characterization of Chondrosin, a New Protein with Anti-tumoral Activity from the Marine Sponge Chondrosia Reniformis Nardo 1847.

Chondrosia reniformis is a common marine demosponge showing many peculiarities, lacking silica spicules and with a body entirely formed by a dense collagenous matrix. In this paper, we have described the identification of a new cytotoxic protein (chondrosin) with selective activity against specific tumor cell lines, from C. reniformis, collected from the Liguria Sea. Chondrosin was extracted and purified using a salting out approach and molecular weight size exclusion chromatography. The cytotoxic fractions were then characterized by two-dimensional gel electrophoresis and mass spectrometry analysis and matched the results with C. reniformis transcriptome database. The procedure allowed for identifying a full-length cDNA encoding for a 199-amino acids (aa) polypeptide, with a signal peptide of 21 amino acids. The mature protein has a theoretical molecular weight of 19611.12 and an IP of 5.11. Cell toxicity assays showed a selective action against some tumor cell lines (RAW 264.7 murine leukemia cells in particular). Cell death was determined by extracellular calcium intake, followed by cytoplasmic reactive oxygen species overproduction. The in silico modelling of chondrosin showed a high structural homology with the N-terminal region of the ryanodine receptor/channel and a short identity with defensin. The results are discussed suggesting a possible specific interaction of chondrosin with the Cav 1.3 ion voltage calcium channel expressed on the target cell membranes.

Bile acids regulate intestinal antigen presentation and reduce graft-versus-host disease without impairing the graft-versus-leukemia effect.

Acute graft-versus-host disease (GvHD) causes significant mortality in patients undergoing allogeneic hematopoietic cell transplantation. Immunosuppressive treatment for GvHD can impair the beneficial graft-versus-leukemia effect and facilitate malignancy relapse. Therefore, novel approaches that protect and regenerate injured tissues without impeding the donor immune system are needed. Bile acids regulate multiple cellular processes and are in close contact with the intestinal epithelium, a major target of acute GvHD. Here, we found that the bile acid pool is reduced following GvHD induction in a preclinical model. We evaluated the efficacy of bile acids to protect the intestinal epithelium without reducing anti-tumor immunity. We observed that application of bile acids decreased cytokine-induced cell death in intestinal organoids and cell lines. Systemic prophylactic administration of tauroursodeoxycholic acid (TUDCA), the most potent compound in our in vitro studies, reduced GvHD severity in three different murine transplantation models. This effect was mediated by decreased activity of the antigen presentation machinery and subsequent prevention of apoptosis of the intestinal epithelium. Moreover, bile acid administration did not alter the bacterial composition in the intestine suggesting that its effects are cell-specific and independent of the microbiome. Treatment of human and murine leukemic cell lines with TUDCA did not interfere with the expression of antigen presentation-related molecules. Systemic T-cell expansion and especially their cytotoxic capacity against leukemic cells remained intact. This study establishes a role for bile acids in the prevention of acute GvHD without impairing the graft-versus-leukemia effect. In particular, we provide a scientific rationale for the systematic use of TUDCA in patients undergoing allogeneic hematopoietic cell transplantation.

Migration arrest of chemoresistant leukemia cells mediated by MRTF-SRF pathway

BackgroundDormant chemotherapy-resistant leukemia cells can survive for an extended period before relapse. Nevertheless, the mechanisms underlying the development of chemoresistance in vivo remain unclear.MethodsUsing intravital bone imaging, we characterized the behavior of murine acute myeloid leukemia (AML) cells (C1498) in the bone marrow before and after chemotherapy with cytarabine.ResultsProliferative C1498 cells exhibited high motility in the bone marrow. Cytarabine treatment impaired the motility of residual C1498 cells. However, C1498 cells regained their migration potential after relapse. RNA sequencing revealed that cytarabine treatment promoted MRTF-SRF pathway activation. MRTF inhibition using CCG-203971 augmented the anti-tumor effects of chemotherapy in our AML mouse model, as well as suppressed the migration of chemoresistant C1498 cells.ConclusionsThese results provide novel insight into the role of cell migration arrest on the development of chemoresistance in AML, as well as provide a strong rationale for the modulation of cellular motility as a therapeutic target for refractory AML.

Halosmysin A, a Novel 14-Membered Macrodiolide Isolated from the Marine-Algae-Derived Fungus Halosphaeriaceae sp.

Halosmysin A, a new 14-membered macrodiolide with an unprecedented skeleton, was isolated from the fungus Halosphaeriaceae sp. OUPS-135D-4, which, in turn, was obtained from the marine algae Sargassum thunbergii. The chemical structure of the macrodiolide was elucidated using 1D and 2D NMR, as well as high resolution fast atom bombardment mass (HRFABMS) spectral analysis. The absolute stereochemistry was determined via chemical derivatization and comparison with a known compound, (6R,11R,12R,14R)-colletodiol. Additionally, halosmysin A was shown to be very potent against murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines, with IC50 values ranging from 2.2 ± 3.1 to 11.7 ± 2.8 μM.

A cyclopeptide and three oligomycin-class polyketides produced by an underexplored actinomycete of the genus Pseudosporangium.

Aside from the well-studied conventional actinomycetes such as Streptomyces, the less investigated genera of actinomycetes also represent a promising source of natural products. Genome mining indicated that members of the underexplored genus Pseudosporangium, from which no secondary metabolites have been reported to date, may harbor the biosynthetic machinery for the formation of novel natural products. The strain RD062863, that is available at a public culture collection, was obtained and subjected to metabolite analysis, which resulted in the discovery of a novel cyclopeptide, pseudosporamide (1), along with three new oligomycin-class polyketides, pseudosporamicins A–C (2–4). The unusual structure of compound 1, featured by a biaryl-bond bridging across a tripeptide scaffold, N-acetyl-ʟ-Tyr-ʟ-Pro-ʟ-Trp, was determined by a combination of spectroscopic analyses, chemical derivatization, ECD calculation, and DFT-based theoretical chemical shift calculation, revealing the presence of an (Sa)-axial chirality around the biaryl bond. Compounds 2–4 lacked hydroxylation on the side chain of the spiroacetal rings, which showed clear contrast to other oligomycin congeners and related polyketides with ring-truncation or expansion. The new macrolides 2–4 displayed potent antimicrobial activity against the Gram-positive bacterium Kocuria rhizohpila and the plant pathogenic fungus Glomerella cingulata. All compounds showed moderate cytotoxicity against P388 murine leukemia cells with IC50 values in the micromolar to submicromolar ranges. These results exemplified the validity of phylogeny-focused strain selection combined with biosynthetic gene-directed genome mining for the efficient discovery of new natural products.

Specific, targetable interactions with the microenvironment influence imatinib-resistant chronic myeloid leukemia

Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.

The effect of drying treatment to metabolite profile and cytotoxic potential of Rhizophora apiculata leaves

Abstract. Sibero MT, Siswanto AP, Pribadi R, Sabdono A, Radjasa OK, Trianto A, Frederick EH, Wijaya AP, Haryanti D, Triningsih DW, Hayuningrat SJ, Igarashi Y. 2020. The effect of drying treatment to metabolite profile and cytotoxic potential of Rhizophora apiculata leaves. Biodiversitas 21: 2180-2187. Coastal communities in Indonesia have utilized Rhizophora spp. leaves as a traditional medicine for many years. The previous studies have succeeded in extracting bioactive compounds from this mangrove after drying treatment, but there is a possibility of the compound decomposition or breakdown. This study aimed to determine the effect of drying treatment on the metabolite profile of R. apiculata leaves which were taken from mangrove forests in Rembang, Central Java. The effect of pre-drying treatment was examined by comparing the metabolites profiles of fresh, oven-dried, and sun-dried leaves crude extracts. Extraction was carried out using maceration method with agitation (110 r.p.m.) for 24 hours in methanol. The metabolite profile was analyzed using high-performance liquid chromatography (HPLC) with diode array detector (DAD) and thin layer chromatography (TLC), while secondary metabolites were studied by phytochemical test. The phytochemical results showed that there were no differences in metabolites in dried and fresh R. apiculata leaves. Crude extract of fresh and oven-dried gave 10 spots on the TLC, while sun-dried crude extract had 9 spots. The one missing spot (Rf value of 0.79) in the sun-dried crude extract might have unstable compounds that are easily degraded or damaged by the sunlight. Moreover, HPLC chromatogram indicated the pre-drying treatment gave alteration to the R. apiculata metabolites that only detected at 400 nm. Cytotoxic assay against P388 murine leukemia cell indicated that oven-dried treatment gave the best anticancer activity with IC50 value of 0.0323 mg/mL.

Design, Synthesis, and Biological Evaluation of Novel C5-Modified Pyrimidine Ribofuranonucleosides as Potential Antitumor or/and Antiviral Agents.

Nucleoside analogues are well-known antitumor, antiviral, and chemotherapeutic agents. Alterations on both their sugar and the heterocyclic parts may lead to significant changes in the spectrum of their biological activity and the degree of selective toxicity, as well as in their physicochemical properties. C5-arylalkynyl-β-D-ribofuranonucleosides 3-6, 3΄-deoxy 12-15, 3΄-deoxy-3΄-C-methyl- β-D-ribofurananucleosides 18-21 and 2΄-deoxy-β-D-ribofuranonucleosides 23-26 of uracil, were synthesized using a one-step Sonogashira reaction under microwave irradiation and subsequent deprotection. All newly synthesized nucleosides were tested for their antitumor or antiviral activity. Moderate cytostatic activity against cervix carcinoma (HeLa), murine leukemia (L1210) and human lymphocyte (CEM) tumor cell lines was displayed by the protected 3΄-deoxy derivatives 12b,12c,12d, and the 3΄-deoxy-3΄-methyl 18a,18b,18c. The antiviral evaluation revealed appreciable activity against Coxsackie virus B4, Respiratory syncytial virus, Yellow Fever Virus and Human Coronavirus (229E) for the 3΄-deoxy compounds 12b,14, and the 3΄-deoxy-3΄-methyl 18a,18c,18d, accompanied by low cytotoxicity. This report describes the total and facile synthesis of modified furanononucleosides of uracil, with alterations on both the sugar and the heterocyclic portions. Compounds 12b,14 and 18a,c,d showed noticeable antiviral activity against a series of RNA viruses and merit further biological and structural optimization investigations.