Primary Mononuclear Cells Research Articles

Ornithine lipid is a partial TLR4 agonist and NLRP3 activator

Gram-negative bacterial lipopolysaccharides (LPSs) trigger inflammatory reactions through Toll-like receptor 4 (TLR4) and prime myeloid cells for inflammasome activation. In phosphate-limited environments, bacteria reduce LPS and other phospholipid production and synthesize phosphorus-free alternatives such as amino-acid-containing lipids like the ornithine lipid (OL). This adaptive strategy conserves phosphate for other essential cellular processes and enhances bacterial survival in host environments. While OL is implicated in bacterial pathogenicity, the mechanism is unclear. Using primary murine macrophages and human mononuclear cells, we elucidate that OL activates TLR4 and induces potassium efflux-dependent nucleotide-binding domain and leucine-rich repeat-containing pyrin protein 3 (NLRP3) activation. OL upregulates the expression of NLRP3 and pro-interleukin (IL)-1β and induces cytokine secretion in primed and unprimed cells. By contrast, in the presence of LPS, OL functions as a partial TLR4 antagonist and reduces LPS-induced cytokine secretion. We thus suggest that in phosphate-depleted environments, OL replaces LPS bacterial immunogenicity, while constitutively present OL may allow bacteria to escape immune surveillance.

Increased mRNA expression for serotonin receptor 1B (HTR1B) is associated with thrombosis in BCR::ABL1-negative myeloproliferative neoplasms.

BCR::ABL1-negative myeloproliferative neoplasms (MPNs) are clonal haematopoietic stem cell disorders characterized by specific driver mutations and an increased risk of both macrothrombosis and microthrombosis. Serotonin receptor type 1B (HTR1B) was found to be expressed by various solid tumours, and also primary bone marrow mononuclear cells from myelodysplastic neoplasm and acute myeloid leukaemia patients, representing a potential therapeutic target. In this study we assessed for the first time the expression levels of HTR1B mRNA in the peripheral blood mononuclear cells (PBMC) of 85 newly diagnosed MPN patients, consisting of 28 polycythemia vera, 25 essential thrombocythemia and 32 primary myelofibrosis cases. Levels of HTR1B expression between MPN subtypes and control group were not significantly different. However, at clinical data examination, it was observed that MPN patients with a recent history of major thrombosis and/or signs of impaired microcirculation exhibited significantly higher HTR1B expression levels compared to non-thrombotic MPNs and control group. Moreover, thrombotic MPN patients had significantly higher HTR1B expression than patients with recent thrombosis and absence of MPN diagnostic criteria. These findings suggest that increased levels of HTR1B expression in PBMC might be associated with thrombosis in MPN patients, but larger studies are needed for confirmation, including testing of the receptor protein expression level.

Advanced Analysis and Validation of a microRNA Signature for Fanconi Anemia.

Some years ago, we reported the generation of a Fanconi anemia (FA) microRNA signature. This study aims to develop an analytical strategy to select a smaller and more reliable set of molecules that could be tested for potential benefits for the FA phenotype, elucidate its biochemical and molecular mechanisms, address experimental activity, and evaluate its possible impact on FA therapy. In silico analyses of the data obtained in the original study were thoroughly processed and anenrichment analysis was employed to identify the classes of genes that are over-represented in the FA-miRNA population under study. Primary bone marrow mononuclear cells (MNCs) from sixFA patients and sixhealthy donors as control samples were employed in the study. RNAs containing the small RNA fractions were reverse-transcribed and real-time PCR was performed in triplicate using the specific primers. Experiments were performed in triplicate.The in-silico analysis reported six miRNAs as likely contributors to the complex pathological spectrum of FA. Among these, three miRNAs were validated by real-time PCR. Primary bone marrow mononuclear cells (MNCs) reported a significant reduction in the expression level of miRNA-1246 and miRNA-206 in the FA samples in comparison to controls.This study highlights several biochemical pathways as culprits in the phenotypic manifestations and the pathophysiological mechanisms acting in FA. A relatively low number of miRNAs appear involved in all these different phenotypes, demonstrating the extreme plasticity of the gene expression modulation. This study further highlights miR-206 as a pivotal player in regulatory functions and signaling in the bone marrow mesenchymal stem cell (BMSC) process in FA. Due to this evidence, the activity of miR-206 in FA deserves specific experimental scrutiny. The results, here presented, might be relevant in the management of FA.

β-Carboline derivatives are potent against Acute Myeloid Leukemia in vitro and in vivo.

β-carboline alkaloids exert a distinguished ability to impair cell growth and induce cell death in a variety of cancers and the evaluation of such new therapeutic candidates may denote new possibilities for leukemia treatment. In this present study, we screened 12 β-carboline derivatives containing different substituents at 1- and 3-positions of β-carboline nucleus for their antineoplastic activities in a panel of leukemia cell lines. The cytotoxic effects of the β-carboline derivatives were evaluated in different leukemia cell lines as well as reactive oxygen species (ROS) generation, autophagy, and important signaling pathways. Treatment with the β-carboline derivatives resulted in a potent antineoplastic activity leading to a reduced cell viability that was associated with increased cell death in a concentration-dependent manner. Interestingly, the treatment of primary mononuclear cells isolated from the peripheral blood of healthy donors with the β-carboline derivatives showed a minor change in cell survival. The antineoplastic activity occurs by blocking ROS production causing consequent interruption of the PI3K/AKT and MAPK/ERK signaling and modulating autophagy processes. Notably, in vivo, AML burden was diminished in peripheral blood and bone marrow of a xenograft mouse model. Our results indicated that β-carboline derivatives have an on-target malignant cell-killing activity and may be promising candidates for treating leukemia cells by disrupting crucial events that promote leukemia expansion and chemotherapy resistance.

Characterization of Nanoprecipitated PET Nanoplastics by 1H NMR and Impact of Residual Ionic Surfactant on Viability of Human Primary Mononuclear Cells and Hemolysis of Erythrocytes.

Manufactured nanoplastic particles (NPs) are indispensable for in vitro and in vivo testing and a health risk assessment of this emerging environmental contaminant is needed. The high surface area and inherent hydrophobicity of plastic materials makes the production of NPs devoid of any contaminants very challenging. In this study, we produced nanoprecipitated polyethylene terephthalate (PET) NPs (300 nm hydrodynamic diameter) with an overall yield of 0.76%. The presence of the ionic surfactant sodium dodecyl sulfate (SDS) was characterized by 1H NMR, where the relative ratio of NP/surfactant was monitored on the basis of the chemical shifts characteristic of PET and SDS. For a wide range of surfactant/NP ratios (17:100 to 1.2:100), the measured zeta potential changed from -42.10 to -34.93 mV, but with an NP concentration up to 100 μg/mL, no clear differences were observed in the cellular assays performed in protein-rich media on primary human cells. The remaining impurities contributed to the outcome of the biological assays applied in protein-free buffers, such as human red blood cell hemolysis. The presence of SDS increased the NP-induced hemolysis by 1.5% in protein-rich buffer and by 7.5% in protein-free buffer. As the size, shape, zeta potential, and contaminants of NPs may all be relevant parameters for the biological effects of NPs, the relative quantification of impurities exemplified in our work by the application of 1H NMR for PET NPs and the ionic surfactant SDS could be a valuable auxiliary method in the quality control of manufactured NPs.

Navtemadlin, a Novel MDM2 Inhibitor, Potentiated Venetoclax-Induced Antitumor Efficacy in TP53 Wild-Type Acute Myeloid Leukemia (AML)

Introduction: The tumor suppressor p53 ( TP53) maintains genomic integrity, and nearly 50% of cancers are characterized by a loss or mutation in the TP53 gene. Murine double minute 2 (MDM2) negatively regulates p53 expression and promotes oncogenesis in TP53 wild-type ( TP53 WT) tumors. Navtemadlin (nvtm) is a potent, selective, orally available MDM2 inhibitor that overcomes MDM2 dysregulation by restoring p53 activity and inducing apoptosis in TP53 WT tumors. AML is a heterogeneous hematological malignancy resulting from the transformation of stem cells and is characterized by poor therapy response. Thus, combination therapies are required to avoid tumor escape mechanisms, such as alterations in apoptotic machinery and metabolism. Venetoclax (ven), a BCL-2 inhibitor, is a promising therapy in AML. Overexpression of anti-apoptotic members of the BCL-2 family, including myeloid cell leukemia-1 (MCL-1) limits ven response, thus requiring MCL-1 inhibitors to further improve sensitivity. A proof-of-concept study with idasanutlin (idasa), a first-generation MDM2 inhibitor and ven demonstrated manageable safety and encouraging efficacy in relapsed/refractory AML patients (Daver et al. Blood 2023). Here, we present a preclinical evaluation of nvtm alone and in combination with ven in the setting of TP53 WT AML, with a focus on metabolic pathways, pro-survival signaling, and bone marrow (BM) niche. Methods: AML-derived cell line, MOLM-13 ( TP53 WT, FLT3-ITD) was cultured alone, with HS5 AML stromal cells or with cytokines. Cultures were treated with DMSO, nvtm (0.1-1.5 μM), or ven (0.5 μM) for short-term (4-8 hrs) or long-term (48-72 hrs) and analyzed for changes in cell cycle progression, viability, clonogenicity potential, and metabolism (glycolysis and mitochondrial oxidative phosphorylation by analyzing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) respectively). Primary AML BM mononuclear cells (MNCs) were cultured in cytokine support media. Results: In MOLM-13 cells, short-termnvtm (0.5 µM) treatment rapidly restored p53 expression and phosphorylation, with greater efficacy than idasa. Long-term treatment significantly induced apoptosis in a dose-dependent manner from 0.25 μM (p<0.01) with 80% cytotoxicity at 0.75 μM (p<0.001). Cell cycle analysis at 48 hr showed that nvtm significantly arrested cells in the sub-G0-G1 phase. Clonogenicity potential was significantly lowered in nvtm-treated cells. Metabolic alterations in the Mito Stress and Glycostress tests showed nvtm significantly lowered EACR, OCR, and mitochondrial membrane potential, as compared to idasa. We next evaluated the effects of nvtm in ven-treated MOLM-13 cells. Combination treatment significantly lowered the metabolism, cell viability, and clonogenicity potential of MOLM-13 cells, compared to nvtm or ven treatment. Immunoblotting analysis revealed a reduction in key anti-apoptosis proteins (BCL-2, MCL-1, PARP). Strikingly, nvtm downregulated ven-induced MCL-1 expression in a dose-dependent manner. Culturing MOLM-13 cells in HS5-conditioned media, transwells overlying HS5 cells, or with HS5 cells increased MOLM-13 cell resistance to nvtm. However, in HS5 co-cultures, a combination of nvtm and ven strongly re-sensitized MOLM-13 cells for apoptosis. Nvtm also significantly blocked the cytoprotective effects of AML-relevant cytokines (IL-6, SCF, FLT3 ligand, TPO, and GM-SCF). In cultures of primary AML BM-MNCs (n=11), nvtm alone at clinically relevant concentrations reduced viability (14-40%), and a combination nvtm and ven significantly enhanced AML cytotoxicity (30-52%). Conclusions: Nvtm, a potent second-generation MDM2 inhibitor, rapidly restored p53 function, induced apoptosis in p53 WT AML MOLM-13 by impeding cell cycle, viability, clonogenicity potential, and reducing both glycolysis and oxidative phosphorylation. Nvtm combined with ven significantly enhanced these effects. Importantly, this combination blocked stromal cell-mediated (contact and soluble factors) cytoprotection. These encouraging results provide a mechanistic basis for the evaluation of this combination in vivo and as a therapeutic strategy to overcome documented ven resistance mechanisms in TP53 WT AML patients.

Targeting Oncogenic Enhancers in ASXL1-Mutant Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with limited therapeutic options and dismal outcomes. Truncating mutations in the epigenetic modifier ASXL1 are common and lead to a proliferative disease phenotype, resistance to epigenetic therapies, and higher rates of AML transformation. ASXL1-mutant CMML is characterized by widespread epigenetic remodeling and up-regulation of key leukemogenic driver genes (e.g. HOXA9 and its co-factor MEIS1). There is a genotype-specific activation of enhancers in ASXL1-mutant CMML that is associated with up-regulation of these leukemogenic driver genes. Here, we test the hypothesis that disrupting the oncogenic interactions at these genotype-specific enhancers can be exploited for therapeutic benefit. Methods: We identified activated enhancers specific to ASXL1-mutant CMML as previously described ( Binder et al. Nat Commun 2022;13(1):1434). To disrupt the E2542605 enhancer, we created unique mapped single guide RNAs (confirmed by BLAT). We generated a ribonucleoprotein complex using the Alt-R CRISPR-Cas9 system (IDT Technologies, Coralville IA). We combined the RNP along with Electroporation Enhancer to 3x10^5 cells of the monocytic cell line U937 in 20uL strip-wells provided by the SF Cell Line 4D-Nucleofector X Kit (Lonza, Basel, Switzerland). To deliver the RNP, U937 cells were electroporated via listed cell line specific protocol by the Amaxa 4D-Nucleofector (Lonza). Forty-eight hours post-electroporation, cells were split and sequenced for E2542605 genomic structure disruption. In parallel, RNA was harvested, converted to cDNA and qPCR for HOXA9 and MEIS1 expression was performed using PrimeTime qPCR FAM labeled probe assays (IDT). Gene expression was determined using delta-delta Ct method. For assessment of selective sensitivity of ASXL1 / enhancer interactions, we performed progenitor colony forming assays using primary bone marrow mononuclear cells from CMML patients with or without ASXL1 mutations and healthy normal controls (n=2 in each category), exposed to increasing concentrations of the dual p300/CBP and BET bromodomain inhibitor EP31670. Briefly, we plated cells in methocellulose fortified with recombinant human cytokines supporting hematopoietic growth in duplicate, in a range of EP31670 concentrations. After 10 days, visible hematopoietic colonies were quantified and characterized. IC 50 values were generated using non-linear regression log(EP31670) vs. normalized response. Results: We computationally identified candidate transcriptional regulators enriched at the activated enhancers specific to ASXL1-mutant CMML, including BRD4 (FDR<2.57x10 -370), CBP (FDR=2.56x10 -217), and p300 (FDR=6.36x10 -93). We selected the ASXL1-mutant-specific enhancer E2542605 (predicted to drive HOXA9 expression), given the up-regulation of HOXA9 in ASXL1-mutant CMML (log 2-fold change +2.05, FDR=0.006, n=14). We disrupted E2542605 using two different CRISPR guides, leading to a PCR trace detection of a 14bp deletion and a 26bp indel in the E2542605 enhancer region, respectively. Both enhancer disruptions led to a down-regulation of its putative target gene HOXA9: Mean relative fold-change 0.36 (p=0.001, n=3) and 0.65 (p=0.046, n=3), respectively ( Fig. 1a). We then used a pharmacologic approach (EP31670) to target the candidate transcriptional regulators involved in the oncogenic interaction at these ASXL1-mutant-specific enhancers. We observed selective sensitivity to growth of CMML colonies (particularly ASXL1-mutant colonies) compared to ASXL1-wild type cases and normal bone marrow controls ( Fig. 1b). Conclusions: In CMML patients, truncating ASXL1 mutations are associated with the activation of genotype-specific enhancers and the recruitment of an extended repertoire of transcriptional regulators including BRD4, CBP, and p300. In a model system, the disruption of an enhancer predicted to govern the expression of the leukemogenic driver HOXA9, led to a significant down-regulation of HOXA9 expression. Pharmacologically disrupting the oncogenic interaction at such enhancers with EP31670 in primary patient samples led to significant therapeutic effects at nanomolar drug concentrations in a genotype-specific manner. Oncogenic interactions at activated enhancers are viable therapeutic targets in ASXL1-mutant CMML and warrant further clinical investigation.

Intracellular persistence of Leishmania tarentolae in primary canine macrophage cells

Leishmania tarentolae is a non-pathogenic species first isolated from geckoes in the Mediterranean basin. The finding that dogs test positive against both Leishmania infantum and L. tarentolae raises questions regarding the ability of the latter species to persist and adapt to new hosts. This study aimed to evaluate in vitro the capability of L. tarentolae to colonize, survive and persist in canine primary monocyte-derived mononuclear cells. Monocytes were isolated from dog whole blood samples and placed in 24-well plates for differentiation into macrophages and for incubation with L. tarentolae field-isolated strains (RI-325 and SF-178) and laboratory (LEM-124) strain; the parasite burden was assessed at different time points post-infection. The L. infantum laboratory strain (MON-1) was used as control. Infection parameters were evaluated by microscopy, counting the number of amastigotes/200 infected cells, and by duplex real-time PCR from supernatants and detached cells. Similar to L. infantum, L. tarentolae strains developed into round-shaped amastigote-like forms, with higher infection rates detected at 4 h followed by an overall decrease until 48 h. RI-325 presented also a higher infection rate at 72 h. Data showed that L. tarentolae strains infect and persist inside in vitro primary canine mononuclear cells, opening new perspectives for further laboratory studies.

Abstract 5150: Improvement of hematopoiesis in primary low risk MDS with the TGFBR1 investigational inhibitor TP-6379

Abstract Background: The inflammaging modulator, S100A9, mediates immunosuppression and plays a key role in the pathogenesis of low-risk myelodysplastic syndrome (MDS). Specifically, S100A9’s feedforward activation in the bone marrow (BM) induces pyroptotic cell death of hematopoietic stem and progenitor cells (HSPC) and the activation and accumulation of myeloid-derived suppressor cells (MDSC). This process creates a suppressive microenvironment including the secretion of transforming growth factor β (TGFβ) that signals through the TGFβ receptor 1 (TGFBR1) to induce inhibitory processes that contribute to this phenotype. We hypothesize that targeting with an investigational TGFBR1 inhibitor, TP-6379, reduces immune suppression and restores hematopoiesis in MDS. Methods: Primary low risk MDS BM mononuclear cells (BMMNC) were obtained from the Moffitt Total Cancer Care Protocol and cultured in vitro with TP-6379. Healthy BMMNC were purchased, treated with or without recombinant S100A9 and TP-6379. Results: Treatment of primary MDS BMMNC (n=15) and S100A9-treated healthy BMMNC (n=5) with TP-6379 for 48 hours was observed to significantly improve proliferation of hematopoietic progenitor cells as measured by colony forming capacity. Total colonies, and BFU-E specific colonies, showed significant increase. This expansion of progenitors with treatment was validated by flow cytometric analysis of Lineage−HLA-DR−CD34+ HSPC and showed a reduction in genomic instability, measured by γH2AX. In addition, TP-6379 was observed to reduce the numbers of suppressive mediators; MDSC, T regs and senescent lymphocytes and increase the number of cytotoxic cells, including NK cells. Our analysis revealed that BMMNC with spliceosomal mutations, including SF3B1, have elevated sensitivity to TP-6379 treatment. Therefore, we tested wild type and SF3B1 K700E CRISPR knock-in K562 cells in the presence of TP-6379 for 48 hours. K562 is a well-established cell line model for study of MDS. The SF3B1 mutant expressing cells have significantly reduced BFU-E colony forming capacity, compared to wild type cells, concordant with higher pyroptosis activation and consistent with the BMMNC results. Hence, we measured phospho-SMAD activity, as a direct measure of TGFBR1 inhibition. SF3B1 mutant expressing cells were shown to be highly sensitive to TP-6379, with a significant loss of SMAD activation. Expression analysis of primary MDS, S100A9-treated healthy BMMNC cells and K562 SF3B1 mutant cells revealed elevated expression of SERPINE1 which was rescued by TP-6379 treatment. Conclusions: TP-6379 restores healthy hematopoiesis in low-risk MDS, which may translate into a strong therapeutic option in a disease with few clinical options. Importantly, our work also suggests a specific role for spliceosome dysfunction in bone marrow failure and a novel role for SERPINE1 in MDS and S100A9-induced suppression. Citation Format: Erika A. Eksioglu, Gabriela M. Wright, Jason M. Foulks, Matthew Lalonde, Steven L. Warner, Kenneth L. Wright. Improvement of hematopoiesis in primary low risk MDS with the TGFBR1 investigational inhibitor TP-6379. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5150.

Design of a novel bioink suitable for the 3D printing of lymphoid cells

Introduction: For decades, in vitro 2D cell culture techniques have been employed in research, but they fail to recapitulate the complexity of natural tissues. 3D bioprinting could potentially overcome this drawback due to the possibility to control the spatial disposition of living cells and the geometry of the 3D scaffold.Materials and methods: This study reports the design and characterization of a novel bioink for extrusion bioprinting, analyzing different blend formulations composed of alginate, gelatin, and methylcellulose, suitable as cell-laden bioink for lymphoid cells, in particular those isolated from patients with Chronic Lymphocytic Leukemia (CLL). The rheological properties as a function of temperature and the printability of the formulations were investigated to define the optimal printing parameters. In vitro stability of the printed scaffolds was investigated under culture conditions and compression tests were performed on printed and bioprinted scaffolds to compare their mechanical properties with those of fresh lymphoid tissue. Finally, MEC1, a CLL cell line, was bioprinted to investigate cell viability, cell density, and cell capability to be released from the scaffold over time.Results and discussion: Results showed that, for the selected blends, good shape fidelity and printing accuracy were achieved with a limitation on the number of printed layers. Scaffolds withstood culture conditions showing stability for up to 3 weeks and their mechanical properties were similar to those of lymphoid tissues already reported in the literature. High cell viability after 21 days was observed for both MEC1 and primary peripheral mononuclear cells, confirming the possibility to use the selected formulation to successfully bioprint lymphoid cells by possibly mimicking their native lymphoid microenvironment.

Metformin Inhibits STAT3 and MAPK Signaling through AMPK Activation in CSF3RT618I Cells

Introduction: In the light of the discovery of the CSF3R mutation in chronic neutrophilic leukemia (CNL) patients, the T618I mutation demonstrated to be a molecular marker and a possible target for new therapies due to the JAK/STAT signaling pathway activation [N Engl J Med (2013) 368(19):1781-90]. Since no curative treatment options are available for patients diagnosed with CNL, it is of interest the study new therapeutical approaches for these patients. In this regard, the treatment of JAK2V617F murine models with metformin, an FDA-approved drug for type 2 diabetes, reduced splenomegaly, extramedullary hematopoiesis, and colony formation of HSPC [Cell Death Dis. 2018;9(3):311]. Hence, we characterized the preclinical cellular and molecular effects of metformin in CSF3RT618I models. Methods: The in vitro experiments were performed in Ba/F3 cells transduced retrovirally with CSF3RT618I overexpression (OE) model. Cells were treated with metformin (1-20 mM) to access cell viability and survival. Protein expression was evaluated by western blot. For in vivo evaluation, eight-week-old female NSG mice were transplanted with 2x106 Ba/F3 CSF3RT618I cells, resuspended in Matrigel: PBS (1:1 ratio). After the tumor formation (2 days), mice were treated for 10 days by intraperitoneal injection of vehicle (PBS) or metformin (125 mg/kg, n=10/group). Tumor volume was measured using the formula (V = W2 × L × 0.52), where W and L are the smallest and largest diameters, respectively. Ex vivo primary cells from a patient diagnosed with CNL harboring the CSF3RT618I mutation were used to assess the effects of metformin treatment. The primary bone marrow mononuclear cells were evaluated regarding cell survival and clonogenic capacity upon metformin treatment. All experiments were approved by the Animal and Human Ethics Committee of the Institution. Statistical analyses were performed by Mann-Whitney or ANOVA test and Bonferroni post-test. A p-value < 0.05 was considered. Results: After 24 and 48 hours, Ba/F3 CSF3RT618I treated with 1, 5, 10 and 20 mM of metformin presented a reduction in cell viability in a dose- and time-dependent manner (p < 0.01). The IC50 for metformin treatment for 24, 48, and 72 hours was 17.44, 1.3, and 3.2 mM, respectively. Increased frequency of apoptotic cells was observed at 5, 10, and 20 mM of metformin at times of 24, 48, and 72 hours (p < 0.01). Molecularly, CSF3RT618I cells displayed reduced STAT3 and ERK phosphorylation and increased AKT/mTOR activation, upon metformin 5 mM treatment for 24 hours. AMPK activation was observed in cells treated with 1-20 mM of metformin compared to vehicle. In vivo, metformin treatment decreased the tumor burden of CSF3RT618I transplanted mice after 9 days of treatment (vehicle versus metformin mean ± SEM: 731.9±90.14 vs. 481.6±38.14; p = 0.035). At the protein level, we observed inhibition in Cyclin D1 and the presence of caspase 3 cleavage in the isolated tumor from the metformin-treated mice. Ex vivo metformin treatment of the primary cells isolated from the CNL patient harboring CSF3RT618I resulted in increased apoptosis, with 51.94% (p < 0.05), 52.40% (p < 0.05), and 63.1% (p < 0.001), of apoptosis induction upon treatment with 5, 10 and 20 mM. Metformin ex vivo treatment strongly reduced hematopoietic progenitor cells colony formation on primary CNL CSF3RT618I peripheral blood (83.5% at 10 mM and 100% at 20 mM) and bone marrow mononuclear cells (100% at 10 mM and 98% at 20 mM). Conclusions: We provide evidence that metformin exerts anticancer activity in CSF3RT816I cells, by downregulation of STAT3 and MAPK signaling through AMPK activation. These results support the idea that metformin can be repurposed in the CNL clinics, as a therapeutical option.

VIP943 Is a Novel CD123 Antibody Drug Conjugate with in Vitro and In Vivo Efficacy in Acute Myeloid Leukemia (AML) Models

Background: Current treatment options (cytarabine with anthracyclines) for patients with acute myeloid leukemia (AML) are often associated with severe and barely tolerable toxicities. In patients who tolerate induction therapy, the complete remission rate is 60%-80%; however, 50% of patients relapse due to the presence of leukemic stem cells (LSC). Antibody-drug conjugates (ADCs) are putatively designed to deliver potent drugs directly to cancer cells while sparing normal tissue; however, first-generation ADCs are associated with severe bone marrow and liver toxicity. VIP943 is a CD123-targeting ADC which uses a kinesin spindle protein inhibitor (KSPi) as a new payload class and a unique legumain linker designed to overcome liabilities of currently approved ADC therapies. The payload is delivered with a drug-antibody ratio of 6. It consists of the KSPi warhead which is modified with a CellTrapperTM moiety so it accumulates within the CD123-targeted tumor cell and cannot enter normal cells. Non-human primate toxicology studies demonstrated a highly favorable and differentiated safety profile for VIP943 (Kirchhoff et al, Cancers 2020). Here we characterize VIP943 in various patient-derived AML models. Methods: For co-localization experiments, MOLM-13 AML cell line was stained with labelled anti-CD123 antibody or isotype control and CytoPainter LysoGreen indicator reagent and analyzed by imaging flow cytometry. AML primary patient mononuclear cells from blood and bone marrow were treated with VIP943 for 24, 48, and 72 hours. Flow cytometry immunophenotyping for AML blasts and CD34+/CD38- LSCs was used to analyze these populations. An in vivo patient-derived AML PDX mouse model was treated with VIP943 (5 mg/kg IV every 7 days) or in combination with 5-azacytidine (2.5 mg/kg SC days 1-5 x 3) and venetoclax (50 mg/kg PO days 1-5 x 3). Cytokine release syndrome was analyzed comparing VIP943 to isotype control ADC, anti-CD123 antibody, and positive comparator antibodies by in vitro cytokine release assays (CRA) in two different assay formats using whole blood and isolated mononuclear cells from 10 healthy donors. Flow cytometric lymphocyte and leukemic immunophenotyping was performed from blood of non-human primates. A leukemia cell line panel drug screen with VIP943 was evaluated and baseline genomic features were correlated with response. Results: Co-localization studies confirm specific internalization and efficient intracellular trafficking to the lysosome of VIP943. In vitro treatment with VIP943 (66 nM, continuous exposure) delivers blast reductions of 37% after a 24h incubation with VIP943, 45% after 48h incubation and 83% after an 72h incubation. In the CD34+/CD38- cell population, a reduction of 71% was achieved. In a patient-derived AML model, VIP943 with 5-azacytidine and venetoclax led to complete tumor growth inhibition after 21 days showing superiority of this triplet therapy (complete regression: 8/9 mice) with high tolerability (maximum body weight loss -4.8 %). The immunotoxicological evaluation in both CRA formats and with the tested antibody concentrations (100µg, 10µg, 1µg) exerts no induction of cytokines by VIP943 compared to controls. Immunophenotyping demonstrates no impact of VIP943 treatment on lymphocytic subpopulations, whereas the CD123-positive basophils are reduced after 24h followed by an almost complete recovery. VIP943 IC50 ranges from 0.1nM-300nM in a leukemia cell line panel screen and features associated with sensitivity are investigated. Conclusions: VIP943 is a next-generation ADC with a differentiated safety profile from currently approved ADCs, including lack of in vitro cytokine release. In vitro and in vivo studies using patient-derived AML cells show VIP943 has favorable monotherapy and combination efficacy including targeting of leukemic stem cells, which drive relapse. These findings warrant evaluating VIP943 in clinical trials.

Novel myeloma patient-derived xenograft models unveil the potency of anlotinib to overcome bortezomib resistance.

Multiple myeloma (MM) remains a common hematologic malignancy with a 10-year survival rate below 50%, which is largely due to disease relapse and resistance. The lack of a simple and practical approach to establish myeloma patient-derived xenograft (PDX) hampers translational myeloma research. Here, we successfully developed myeloma PDXs by subcutaneous inoculation of primary mononuclear cells from MM patients following series tumor tissue transplantations. Newly established myeloma PDXs retained essential cellular features of MM and recapitulated their original drug sensitivities as seen in the clinic. Notably, anlotinib therapy significantly suppressed the growth of myeloma PDXs even in bortezomib-resistant model. Anlotinib treatments polarized tumor-associated macrophages from an M2- to an M1-like phenotype, decreased tumor vascular function, and accelerated cell apoptosis in myeloma PDXs. Our preclinical work not only unveiled the potency of anlotinib to overcome bortezomib resistance, but also provided a more practical way to establish MM PDX to facilitate myeloma research.

Vaccine mRNA Can Be Detected in Blood at 15 Days Post-Vaccination.

COVID-19 mRNA vaccines effectively reduce incidence of severe disease, hospitalisation and death. The biodistribution and pharmacokinetics of the mRNA-containing lipid nanoparticles (LNPs) in these vaccines are unknown in humans. In this study, we used qPCR to track circulating mRNA in blood at different time-points after BNT162b2 vaccination in a small cohort of healthy individuals. We found that vaccine-associated synthetic mRNA persists in systemic circulation for at least 2 weeks. Furthermore, we used transmission electron microscopy (TEM) to investigate SARS-CoV-2 spike protein expression in human leukemic cells and in primary mononuclear blood cells treated in vitro with the BNT162b2 vaccine. TEM revealed morphological changes suggestive of LNP uptake, but only a small fraction of K562 leukemic cells presented spike-like structures at the cell surface, suggesting reduced levels of expression for these specific phenotypes.

Abstract 5492: Activity of RP7214, a novel, selective, and potent small molecule inhibitor of DHODH, in AML

Abstract Background: Dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme in pyrimidine synthesis, is overexpressed in certain cancers and impedes tumor cell proliferation via cell cycle arrest. RP7214 is a novel and potent DHODH inhibitor that inhibited DHODH activity in enzyme and PHA induced HWB/PBMC proliferation with IC50 and EC50 values of 7.8 &amp; 2.5/0.60 nM respectively. Herein, we describe the efficacy of RP7214 as a single agent and in combination with standard of care drugs in preclinical models of AML Methods: Cell viability of AML cell lines (U937, HL-60, THP-1, and KG-1) following incubation with RP7214 as a single agent or in combination with Azacytidine or Venetolcax for 72 h was determined in an MTT-based assay. Cell surface expression of CD11b in THP-1 and human primary bone marrow AML mononuclear cells in presence of RP7214 was evaluated by flow cytometry. Apoptosis was evaluated following incubation of cell lines with compound for 48 or 72 h, subsequent staining with Annexin-V-PE and 7-AAD, and analysis by flow cytometry. Anti-tumor potential of RP7214 as single agent was tested in subcutaneous MV-4-11 human leukemia xenograft model. Results: RP7214 inhibited AML cell proliferation in all the four cell lines tested with GI50 ranging from 2.4 to 7.6 µM. Expression of the CD11b marker on THP-1 cell surface was enhanced by 40% following incubation with 5 µM compound; effect of which was reduced to 15% in the presence of 300 µM Uridine. Incubation of primary human bone marrow AML mononuclear cells with 10 µM of RP7214 increased the CD11b+ cells by &amp;gt; 15 %. Combination of 3 µM-RP7214 and 1 µM-Azacytidine inhibited cell proliferation by &amp;gt; 25% (P &amp;lt; 0.05) compared to single agents in MV-4-11 cells. Similarly, combination of 3 µM-RP7214 with 3 nM Venetoclax demonstrated 15% (P &amp;lt; 0.05) inhibition of cell growth compared to single agents in MV-4-11 cell line. RP7214 at 3, 10, and 30 mg/kg BID demonstrated significant anti-tumor activities in both tumor size and tumor weight in subcutaneous MV-4-11 human leukemia xenograft model. Conclusions: Results demonstrate the utility of RP7214 as a single agent and in combination with standard of care drugs in inducing apoptosis and promoting differentiation to fully mature cells in AML. A Phase 2a trial in AML patients is planned. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Swaroop Vakkalanka. Activity of RP7214, a novel, selective, and potent small molecule inhibitor of DHODH, in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5492.

A Comparative Study on the Viability of Normal and Cancerous Cells upon Irradiation with a Steady Beam of THz Rays

Terahertz (THz) electromagnetic radiation is commonly used in astronomy, security screening, imaging, and biomedicine, among other applications. Such approach has raised the question of the influence of THz irradiation on biological objects, especially the human body. However, the results obtained to date are quite controversial. Therefore, we performed a comparative study on the viability of normal cells and cancer cells upon irradiation with a steady beam of THz rays. We used human peripheral blood mononuclear cells and cancer cell lines. Primary human mononuclear blood cells (monocytes, and B-, and T-cells) showed an increased death rate, determined by cell counting and fluorescence microscopy, upon 0.14 THz irradiation. The effect of THz radiation was different among malignant cells of B- and T-cell origin (Ramos and Jurkat cells) and epithelial cancer cells (MCF7 and LNCaP). This was demonstrated by cell counting and by the alamarBlue assay. In conclusion, THz radiation can result in the death of human primary and malignant cells. However, the mechanism of this phenomenon is largely unknown. Hence, more work should be done to shed some light on the mechanism of action of THz irradiation in living organisms to enhance technologic developments.

Selected Articles from This Issue

Despite previous assumptions that constitutively high Wnt signaling promotes colorectal cancer in each stage of disease, recent studies have demonstrated that reduced Wnt signaling is associated with colorectal cancer invasiveness and poor prognoses. However, little work has been done to elucidate how diminished Wnt signaling underlies aggressive colorectal cancer phenotypes. Chen and colleagues used genetic manipulation strategies and orthotopic mouse models to show that abrogating Wnt signaling increases colorectal cancer invasiveness and decreases immune responsiveness in vivo. Disrupting Wnt signaling by targeting Wnt coreceptor LDL receptor-related protein 6 (LRP6) with CRISPR/Cas9 or transducing a dominant negative version of Wnt transcriptional effector lymphoid enhancer binding factor 1 (LEF1) increases tumor submucosal invasion and dissemination in mice. Using RNA-Seq, the authors revealed that reducing Wnt signaling elevates expression of markers associated with stemness and a mesenchymal phenotype, while repressing expression of immune-stimulatory molecules and collagens. The authors used TCGA data to demonstrate that the low Wnt signaling expression profile corresponds with poor survival in colorectal cancer patients, underscoring the potential clinical relevance of dampened Wnt signaling in colorectal cancer progression.Cholangiocarcinoma (CCA) is an aggressive biliary tract cancer for which effective therapeutics are lacking. Prostaglandin E2 (PGE2)-mediated inflammation is a critical component of CCA carcinogenesis and progression. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) chemically modifies and inactivates PGE2, but its expression is often diminished in CCA. In their work, Zhang and colleagues leveraged public data to show that expression of histone methyltransferase G9a negatively correlates with 15-PGDH expression. The authors validated the G9a-15-PGDH relationship in a mouse model of CCA in which the Notch intracellular domain and constitutively active Akt are expressed via hydrodynamic injection of sleeping beauty transposase-based plasmids. shRNA-mediated G9a silencing increases 15-PGDH expression in CCA cell lines, limiting cell proliferation in vitro and tumor growth in vivo. ChIP assays demonstrated that G9a is recruited to the 15-PGDH promoter by the Myc/Max/E-box complex, which prevents signal transducer and activator of transcription 4 (STAT4) from binding the promoter and inducing 15-PGDH expression. The work presented elucidates a previously unknown mechanism underlying CCA development and progression and reveals a novel potential target for CCA therapeutic strategies.Neurofibromatosis type 2 is characterized by growth of benign nervous system tumors, such as schwannomas, which can lead to deafness, brain stem compression, and death. Neurofibromatosis type 2 is caused by mutations in tumor suppressor gene NF2. Previously, Cho and colleagues demonstrated that NF2 loss promotes elevated TβR1 signaling, resulting in phosphorylation and degradation of tumor suppressor Raf kinase inhibitor protein (RKIP). While TGF-β inhibitors halt schwannoma growth, they are not ideal therapeutics given schwannomas occur around adolescence and TGF-β is required for development. Here, Cho and colleagues developed a novel TβR1 inhibitor, Nf18001, that abrogates TβR1-mediated RKIP degradation while maintaining canonical TGF-β signaling. The authors used human and mouse schwannoma cell lines to demonstrate that Nf18001 disrupts TβR1-RKIP interactions to sustain RKIP expression and lower NF2-mutant cell viability while not influencing normal fibroblast survival. Nf18001 arrests cell cycle progression in schwannoma cells, and augments Schwann cell differentiation marker expression while diminishing expression of markers associated with cell stemness. Nf18001 also displayed efficacy in a schwannoma allograft mouse model, where treatment slowed tumor growth.The pro-tumorigenic role of signal transducer and activator of transcription 3 (STAT3) in multiple myeloma has been established, but therapeutically targeting STAT3 has proven challenging. Similarly, given its canonical role in DNA damage repair, checkpoint kinase 1 (Chk1) inhibition has been tested in anti-cancer therapeutic regimens, but with ambiguous results. In their study, Zhou and colleagues employed a gene expression array to demonstrate that Chk1 inhibition diminishes expression of oncogenic STAT3 target genes in multiple myeloma cells, suggesting Chk1 inhibition may also effectively inhibit STAT3 activity. Subsequent work showed that Chk1 directly binds to STAT3 and phosphorylates it at tyrosine 705, enabling STAT3 dimerization and DNA binding. Chk1 inhibition — pharmacologically and via shRNA silencing — diminishes STAT3 tyrosine 705 phosphorylation and inhibits STAT3 DNA binding, reducing expression of STAT3 transcriptional targets. Chk1 inhibition abrogates STAT3 tyrosine 705 phosphorylation in IL-6–dependent, -independent, and bortezomib-resistant cells, as well as in patient primary myeloma mononuclear cells and multiple myeloma cells implanted into mice, suggesting Chk1 inhibition may provide therapeutic efficacy against multiple myeloma through a STAT3-related mechanism.

Supplemental aerobic, anaerobic and strength training positively affects autonomic functioning, anaerobic capacity, and immune cell homeostasis of male judo athletes

The present study aimed to investigate the effect of additional package of aerobic, anaerobic and strength training (AAS training), along with the conventional judo specific training, on autonomic nervous system balance, anaerobic capacity, exercise adaptation, phenotype of different blood mononuclear cells and cellular inflammatory signalling of university judo athletes. Thirty male judokas were randomly allocated to a control group/conventional judo specific training group (performing Uchi-komi, Nage komi, and Randori) or to an experimental group/conventional judo specific training + additional AAS training group. Physiological data including recovery ANS variables and anaerobic capacity were obtained by using wireless heart rate variability and anaerobic cycle ergometer, respectively, at two different time points (T1-pre, T3-post). Serum and primary mononuclear cells were prepared at three different time points (T1-pre, T2-acute, T3-post) and processed further as per the experimental requirement. For measuring the expression level of genes, and proteins biomarkers related to immune health, we have performed advanced qPCR array technique, flow cytometry, ELISA and zymosan-fluorescein assays. The additional training modality enhanced athletes’ anaerobic performance, parasympathetic functioning, and exercise adaptation. On the other hand, it decreased fatigue index, stress index, number of reactive immune cells, and intensity of inflammatory signalling. Overall, the present study, for the very first time, exhibited the positive effect of four weeks long additional AAS training on autonomic functioning, anaerobic capacity, and immune cell homeostasis of male judo athletes. This additional training package might also help the judo coaches to optimise training schedule for the competitive session.

Pharmacological characterization of a novel metal-based proteasome inhibitor Na-AuPT for cancer treatment.

The ubiquitin-proteasome system (UPS) is essential for maintaining cell homeostasis by orchestrating the protein degradation, but is impaired in various diseases, including cancers. Several proteasome inhibitors, such as bortezomib, are currently used in cancer treatment, but associated toxicity limits their widespread application. Recently metal complex-based drugs have attracted great attention in tumor therapy; however, their application is hindered by low water-solubility and poor absorbency. Herein, we synthesized a new type of gold (I) complex named Na-AuPT, and further characterized its anticancer activity. Na-AuPT is highly water-soluble (6 mg/mL), and it was able to potently inhibit growth of a panel of 11 cancer cell lines (A549, SMMC7721, H460, HepG2, BEL7402, LNCap, PC3, MGC-803, SGC-7901, U266, and K562). In A549 and SMMC7721 cells, Na-AuPT (in a range of 2.5-20 μM) inhibited the UPS function in a dose-dependent fashion by targeting and inhibiting both 20 S proteasomal proteolytic peptidases and 19 S proteasomal deubiquitinases. Furthermore, Na-AuPT induced caspase-dependent apoptosis in A549 and SMMC7721 cells, which was prevented by the metal chelator EDTA. Administration of Na-AuPT (40 mg · kg-1 · d-1, ip) in nude mice bearing A549 or SMMC7721 xenografts significantly inhibited the tumor growth in vivo, accompanied by increased levels of total ubiquitinated proteins, cleaved caspase 3 and Bax protein in tumor tissue. Moreover, Na-AuPT induced cell death of primary mononuclear cells from 5 patients with acute myeloid leukemia ex vivo with an average IC50 value of 2.46 μM. We conclude that Na-AuPT is a novel metal-based proteasome inhibitor that may hold great potential for cancer therapy.

Mannose Metabolism Is a Metabolic Vulnerability Unveiled By Standard and Novel Therapies in Acute Myeloid Leukemia

IntroductionThe development of resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML). Metabolic rewiring has emerged as a therapeutically actionable vulnerability in AML, where specific metabolic adaptations arising as a result of driver mutations or in response to therapy have been reported. Mannose phosphoisomerase (MPI) is the first enzyme in the mannose metabolism (MM) pathway, that leads to the production of GDP-Mannose, a key sugar donor for N-glycosylation reactions. MPI was amongst the top drop-out genes in our published CRISPR-Cas9 screen aiming to identify sensitizers to FLT3 tyrosine kinase inhibitors (TKI) in AML carrying activating FLT3 internal tandem duplication (ITD) mutations (Gallipoli et al., Blood 2018). Moreover, analysis of published genomic datasets indicates that MPI expression levels are higher in AML compared to normal samples, correlate with patient outcome (A) and further increase in paired relapsed to diagnosis samples. We therefore hypothesized that MM and MPI inhibition sensitize AML cells to both FLT3-TKI and standard chemotherapy and tested this in preclinical models.MethodsExperiments were performed in human AML cell lines, primary AML mononuclear cells and normal CD34 + stem/progenitor cells. Liquid chromatography coupled to mass spectrometry and oxygen consumption/extracellular acidification rate as measured by a Seahorse analyser were employed to assess metabolic changes. Gene-expression was measured by RNA-sequencing and confirmed by RT-qPCR. Viability, surface protein expression and lipid peroxidation were assessed by flow-cytometry. Protein expression and localisation was measured by western blot and immunofluorescence. Gene silencing was performed using CRISPR-Cas9 gene editing and inducible short hairpin RNA interference.ResultsWe show that genetic and chemical MPI inhibition sensitizes FLT3 WT and FLT3 ITD AML cell lines in vitro to standard cytarabine chemotherapy and FLT3-TKI respectively and these effects are rescued by the addition of MPI downstream product mannose. We validate these findings in vivo using MPI knock-out (KO) cell line xenografts and in vitro using primary AML samples following MPI knock-down (B). We also demonstrate a lack of toxicity to normal CD34 + cells.Surprisingly, global metabolomic analysis show that MPI KO cells accumulate fatty acids and particularly polyunsaturated fatty acids (PUFA) (C), due to both increased uptake and reduced fatty acid oxidation (FAO). MPI KO cells downregulate several FAO metabolism genes and this is corroborated by the strong positive correlation of MPI expression levels with multiple genes involved in FAO across multiple primary AML datasets. Metabolic profiling demonstrates that MPI KO cells have reduced oxidative phosphorylation metabolism and in particular are unable to oxidise palmitate, an effect rescued by mannose or the FAO activator fenofibrate (D).We link this metabolic defect to the specific activation of the ATF6 arm of the unfolded protein response (UPR) due to a reduction in protein glycosylation in MPI KO cells. We validate this by demonstrating that activating or inhibiting ATF6 respectively phenocopies or rescues the effects of MPI KO (E) and show this to be secondary to ATF6 driving FAO inhibition, via transcriptional downregulation of PPARα, a master regulator of lipid catabolism.Finally, we show that accumulation of PUFA in MPI KO cells is accompanied by reduced cysteine levels and increased markers of oxidative stress and lipid peroxidation, such as 4-hydroxynonenal (4-HNE), all features associated with ferroptosis (F). We validate this by showing that combining FLT3-TKI with MPI KO leads to ferroptotic cell death in AML cells, which can be rescued by the radical-trapping antioxidant ferrostatin.ConclusionsWe provide further evidence to support the role of metabolic rewiring in driving therapy resistance in AML and show for the first time that targeting MPI and MM sensitizes AML cells to cytarabine and FLT3-TKI. Mechanistically we unveil a novel connection between MM and fatty acid metabolism, via activation of the ATF6 arm of the UPR, leading to cellular PUFA accumulation, lipid peroxidation and ferroptotic cell death. Finally, our findings also suggest that triggering ferroptosis can be leveraged as a therapeutically actionable mechanism driving cell death in therapy-resistant AML cells. [Display omitted] DisclosuresVassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.