Primary Patient Cells Research Articles

Developmental interplay between transcriptional alterations and a targetable cytokine signaling dependency in pediatric ETO2::GLIS2 leukemia

BackgroundSeveral fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia (AML) are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, associated with dismal prognosis.MethodsWe created novel ETO2::GLIS2 models of leukemogenesis through lentiviral transduction and CRISPR-Cas9 gene editing of human fetal and post-natal hematopoietic stem/progenitor cells (HSPCs), performed in-depth characterization of ETO2::GLIS2 transformed cells through multiple omics and compared them to patient samples. This led to a preclinical assay using patient-derived-xenograft models to test a combination of two clinically-relevant molecules.ResultsWe showed that ETO2::GLIS2 expression in primary human fetal CD34+ hematopoietic cells led to more efficient in vivo leukemia development than expression in post-natal cells. Moreover, cord blood-derived leukemogenesis has a major dependency on the presence of human cytokines, including IL3 and SCF. Single cell transcriptomes revealed that this cytokine environment controlled two ETO2::GLIS2-transformed states that were also observed in primary patient cells. Importantly, this cytokine sensitivity may be therapeutically-exploited as combined MEK and BCL2 inhibition showed higher efficiency than individual molecules to reduce leukemia progression in vivo.ConclusionsOur study uncovers an interplay between the cytokine milieu and transcriptional programs that extends a developmental window of permissiveness to transformation by the ETO2::GLIS2 AML fusion oncogene, controls the intratumoral cellular heterogeneity, and offers a ground-breaking therapeutical opportunity by a targeted combination strategy.

Lipopolymer/siRNA Nanoparticles Targeting the Signal Transducer and Activator of Transcription 5A Disrupts Proliferation of Acute Lymphoblastic Leukemia.

The therapeutic potential of small interfering RNAs (siRNAs) in gene-targeted treatments is substantial, but their suboptimal delivery impedes widespread clinical applications. Critical among these is the inability of siRNAs to traverse the cell membranes due to their anionic nature and high molecular weight. This limitation is particularly pronounced in lymphocytes, which pose additional barriers due to their smaller size and scant cytoplasm. Addressing this, we introduce an innovative lipid-conjugated polyethylenimine lipopolymer platform, engineered for delivery of therapeutic siRNAs into lymphocytes. This system utilizes the cationic nature of the polyethylenimine for forming stable complexes with anionic siRNAs, while the lipid component facilitates cellular entry of siRNA. The resulting lipopolymer/siRNA complexes are termed lipopolymer nanoparticles (LPNPs). We comprehensively profiled the efficacy of this platform in human peripheral blood mononuclear cells (PBMCs) as well as in vitro and in vivo models of acute lymphoblastic leukemia (ALL), emphasizing the inhibition of the oncogenic signal transducer and activator of transcription 5A (STAT5A) gene. The lipopolymers demonstrated high efficiency in delivering siRNA to ALL cell lines (RS4;11 and SUP-B15) and primary patient cells, effectively silencing the STAT5A gene. The resultant gene silencing induced apoptosis and significantly reduced colony formation in vitro. Furthermore, in vivo studies showed a significant decrease in tumor volumes without causing substantial toxicity. The lipopolymers did not induce the secretion of proinflammatory cytokines (IL-6, TNF-α, and INF-γ) in PBMCs from healthy volunteers, underscoring their immune safety profile. Our observations indicate that LPNP-based siRNA delivery systems offer a promising therapeutic approach for ALL in terms of both safety and therapeutic efficacy.

Cytidine deaminase-dependent mitochondrial biogenesis as a potential vulnerability in pancreatic cancer cells

Cytidine deaminase (CDA) converts cytidine and deoxycytidine into uridine and deoxyuridine as part of the pyrimidine salvage pathway. Elevated levels of CDA are found in pancreatic tumors and associated with chemoresistance. Recent evidence suggests that CDA has additional functions in cancer cell biology. In this work, we uncover a novel role of CDA in pancreatic cancer cell metabolism. CDA silencing impairs mitochondrial metabolite production, respiration, and ATP production in pancreatic cancer cells, leading to a so-called Pasteur effect metabolic shift towards glycolysis. Conversely, we find that CDA expression promotes mitochondrial biogenesis and oxidative phosphorylation, independently of CDA deaminase activity. Furthermore, we observe that patient primary cells overexpressing CDA are more sensitive to mitochondria-targeting drugs. Collectively, this work shows that CDA plays a non-canonical role in pancreatic cancer biology by promoting mitochondrial function, which could be translated into novel therapeutic vulnerabilities.

Targeting sphingolipid metabolism in chronic lymphocytic leukemia

Elevated levels of circulating C16:0 glucosylceramides (GluCer) and increased mRNA expression of UDP-glucose ceramide glycosyltransferase (UGCG), the enzyme responsible for converting ceramides (Cer) to GluCer, represent unfavorable prognostic markers in chronic lymphocytic leukemia (CLL) patients. To evaluate the therapeutic potential of inhibiting GluCer synthesis, we genetically repressed the UGCG pathway using in vitro models of leukemic B cells, in addition to UGCG pharmacological inhibition with approved drugs such as eliglustat and ibiglustat, both individually and in combination with ibrutinib, assessed in cell models and primary CLL patient cells. Cell viability, apoptosis, and proliferation were evaluated in vitro, and survival and apoptosis were examined ex vivo. UGCG inhibition efficacy was confirmed by quantifying intracellular sphingolipid levels through targeted lipidomics using mass spectrometry. Other inhibitors of sphingolipid biosynthesis pathways were similarly assessed. Blocking UGCG significantly decreased cell viability and proliferation, highlighting the oncogenic role of UGCG in CLL. The efficient inhibition of UGCG was confirmed by a significant reduction in GluCer intracellular levels. The combination of UGCG inhibitors with ibrutinib demonstrated synergistic effect. Inhibitors that target alternative pathways within sphingolipid metabolism, like sphingosine kinases inhibitor SKI-II, also demonstrated promising therapeutic effects both alone and when used in combination with ibrutinib, reinforcing the oncogenic impact of sphingolipids in CLL cells. Targeting sphingolipid metabolism, especially the UGCG pathway, represents a promising therapeutic strategy and as a combination therapy for potential treatment of CLL patients, warranting further investigation.

Boanmycin induces apoptosis and overcomes venetoclax resistance in acute myeloid leukemia

Abstract Objectives This study aimed to investigate the efficacy of boanmycin, a clinical drug used for head and neck cancers, in the treatment of acute myeloid leukemia (AML), particularly in venetoclax-resistant AML cells. Methods The cell viability assay was conducted to measure the inhibitory effects of boanmycin on the AML cell lines and patient primary cells using the CCK8 reagent. The colony formation assay was performed to evaluate the colony formation ability of HL60 and venetoclax-resistant HL60 (HL60-res) cells with or without boanmycin treatment. Flow cytometry was performed to detect cell apoptosis level, and Western blot was used to assess changes in apoptosis-related proteins. Results Our findings reveal that boanmycin significantly inhibits AML cell proliferation and colony formation, and induces apoptosis. Importantly, boanmycin exhibits substantial inhibitory effects on venetoclax-resistant cells, and suppresses the proliferation of peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMMCs) derived from newly diagnosed and relapsed AML patients. Conclusions Boanmycin may overcome venetoclax resistance and offer therapeutic benefits for patients with venetoclax-resistant AML.

Targeting HMGCS1 restores chemotherapy sensitivity in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a common hematological malignancy with overall poor prognosis. Exploring novel targets is urgent and necessary to improve the clinical outcome of relapsed and refractory (RR) AML patients. Through clinical specimens, animal models and cell-level studies, we explored the specific mechanism of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMGCS1) in AML and the mechanism of targeting HMGCS1 to attenuate cell proliferation, increase chemotherapy sensitivity and improve the occurrence and development of AML. Here, we reveal that HMGCS1 is overexpressed in RR patients and negatively related to overall survival (OS). Knocking out HMGCS1 in AML cells attenuated cell proliferation and increased chemotherapy sensitivity, while stable overexpression of HMGCS1 had the opposite effects. Mechanistically, we identified that knockout of HMGCS1 suppressed mitogen-activated protein kinase (MAPK) pathway activity, while overexpression of HMGCS1 could remarkably enhance the pathway. U0126, a MEK1 inhibitor, offset the effects of HMGCS1 overexpression, indicating that HMGCS1 promotes RR AML through the MAPK pathway. Further, we verified that hymeglusin, a specific inhibitor of HMGCS1, decreases cell growth both in AML cell lines and primary bone marrow cells of AML patients. Furthermore, combination of hymeglusin and the common chemotherapeutic drug cytarabine and adriamycin (ADR) had synergistic toxic effects on AML cells. Our study demonstrates the important role of HMGCS1 in AML, and targeting this protein is promising for the treatment of RR AML.

EPEN-27. GAINING A CLUE FROM 1Q: A HIGH-RISK CHROMOSOMAL GAIN IN PEDIATRIC POSTERIOR FOSSA EPENDYMOMA

Abstract BACKGROUND Ependymomas are neuroepithelial tumours that can be classified into distinct molecular subgroups, each exhibiting a unique clinical outcome. Therapeutic efforts against the most aggressive and abundant subgroup of pediatric ependymoma (posterior fossa group A, PFA) have traditionally been hampered by a lack of known recurrent driver mutations. However, approximately 25% of PFA tumours exhibit gains of the chromosome arm 1q which is associated with an extremely poor prognosis despite aggressive treatment. By interrogating the multi-omic landscape of PFAs harbouring this copy number variation, we aimed to uncover genetic dependencies from which actionable therapeutics could be derived. METHODS Transcriptomic (bulk/single-cell RNA sequencing), proteomic (LC-MS/MS) and phosphoproteomic analyses were conducted on patient tissues and primary patient-derived cell lines to gain insight into the pathways that drive 1q gain pathogenesis. Chromatin immunoprecipitation sequencing of H3K27me3 and H3K27ac marked histones was carried out to profile the epigenetic landscape of these tumours, and whole genome CRISPR knockout screens were performed to reveal the contribution of essential genes unique to tumours harboring 1q gains. RESULTS Through the comparison of 1q gain and balanced PFA samples, our multi-omic analyses identified the up-regulation and essentiality of known glioma oncogenic drivers previously uncharacterized in PFA. The investigation of essential candidate genes showed a convergence on pathways related to ciliogenesis, with genetic vulnerabilities informing in vitro drug screening. Candidate genes were further validated by in vivo knockdown using established xenograft mouse models for pre-clinical testing. CONCLUSIONS This combined approach has allowed for the derivation of a functional cancer signature underlying 1q gains in PFA and presents novel therapeutic targets for this high-risk subgroup. PFA ependymoma is a deadly malignancy whose complex biology has so far eluded therapy, and new insights from this project will help to identify the first effective therapies for this vulnerable patient population.

Interleukin-1–mediated hyperinflammation in XIAP deficiency is associated with defective autophagy

AbstractDeficiency of X-linked inhibitor of apoptosis protein (XIAP) is a rare genetic condition that can present with recurrent episodes of hemophagocytic lymphohistiocytosis (HLH), though the exact mechanisms leading to this hyperinflammatory disorder are unclear. Understanding its biology is critical to developing targeted therapies for this potentially fatal disease. Here, we report on a novel multiexonic intragenic duplication leading to XIAP deficiency with recurrent HLH that demonstrated complete response to interleukin (IL)-1β blockade. We further demonstrate using both primary patient cells and genetically modified THP-1 monocyte cell lines that, contrary to what has previously been shown in mouse cells, XIAP-deficient human macrophages do not produce excess IL-1β when stimulated under standard conditions. Instead, nucleotide-binding oligomerization domain–like receptor family pyrin domain containing 3 (NLRP3) inflammasome–mediated hyperproduction of IL-1β is observed only when the XIAP-deficient cells are stimulated under autophagy-promoting conditions and this correlates with defective autophagic flux as measured by decreased accumulation of the early autophagy marker LC3-II. This work, therefore, highlights IL-1β blockade as a therapeutic option for patients with XIAP deficiency experiencing recurrent HLH and identifies a critical role for XIAP in promoting autophagy as a means of limiting IL-1β–mediated hyperinflammation during periods of cellular stress.

Evaluation of lipopolymer as therapeutic siRNA delivery agents in non-malignant and malignant primary cells.

e15189 Background: Targeting leukemia-driving oncogenes via short interfering RNA (siRNA) offers an alternative approach to leukemia treatment that can overcome the limitations of current therapies. Low molecular weight polyethyleneimine grafted with lipids (lipopolymers) have emerged as promising carriers for siRNA delivery to leukemia cells. Lipopolymers harboring select lipids were recently shown to downregulate target oncogene leading to significant anti-leukemic activity in both in vitro and in vivo cell line-based leukemia models. The goal of this study was to evaluate these lipopolymers in (i) PBMCs obtained from healthy donors to determine the potential impact on non-malignant cells, and (ii) primary leukemia patient cells to assess therapeutic efficacy. Methods: Target gene levels were determined by RT-qPCR and the effect of treatment on progenitor cells was assessed by measuring changes in colony forming ability. Results: At day 1 post-treatment, no changes in FLT3 mRNA levels were observed in all 6 PBMC samples with the 2 candidate lipopolymers (LeuFectB and LeuFectC) tested. Among 11 PBMC samples evaluated at day 2, only one sample showed a significant reduction in oncogene transcript levels on treatment with LeuFectB. The impact of lipopolymer/siRNA complex treatment on progenitor cells in the PBMC population was then assessed. Of the 7 PBMC samples tested, significant reduction in colony numbers was observed in one sample with LeuFectB and two samples with LeuFectC. On an average, the lipopolymer/siRNA complexes were well tolerated by normal healthy cells as changes observed in oncogene expression and colony forming ability were not consistent across the whole pool. Primary acute lymphoblastic leukemia (ALL) patient cells treated with complexes targeting STAT5A showed marked reduction in STAT5A transcript levels in 4 out of 7 samples with both the lipopolymers tested. The subsequent impact of STAT5A downregulation on the clonogenicity of leukemia stem cells was observed in the significant decrease in colony numbers seen in 3 samples (out of 8) with LeuFects. Additionally, combined downregulation of STAT5A and BCR-ABL genes in BCR-ABL+ ALL patient samples led to a notable decrease in cell proliferation and viability. Moreover,2 out of 3 acute myeloid leukemia (AML) patient samples harboring the FLT3-ITD mutation showed significant retardation in their colony forming ability on treatment with FLT3-targeting siRNA complexes. Collectively, the ALL and AML primary samples demonstrated promising preliminary results, suggesting a therapeutic effect on the stem cell population which is critical for mitigating relapse or prolonging remission. Conclusions: These findings provide further proof-of-concept for clinical translation of lipopolymer mediated siRNA therapy of leukemia, showing relative safety and selectivity of these lipopolymer/siRNA complexes.

Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML

AbstractThe overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Abstract 7227: Isatin analogs potentiate the cytotoxicity of venetoclax in acute myeloid leukemia cells

Abstract Acute Myeloid Leukemia (AML) is a rare but common hematological malignancy in children and adults. AML is a highly heterogeneous disease caused by the acquisition of several mutations affecting multiple cellular processes, thus requiring concomitant targeting of key survival nodes to treat effectively. As current first-line therapies for AML suffer with poor outcomes, drug resistance, and high relapse rates, there is an immediate need for new combinatorial therapies that are both safe and effective. Venetoclax (VEN), a selective inhibitor of antiapoptotic protein B-cell lymphoma-2 (BCL2), has been FDA-approved for treating adult AML and displayed potential in pediatric leukemia treatment. However, the activation of an antiapoptotic BCL2 family member, Myeloid Cell Leukemia 1 (MCL1), is often a primary mechanism of resistance to VEN therapy. Our group has developed Isatin analogs (ISA) that exhibited diverse biological activities, including anti-cancer activities alone or in combination with standard agents. ISA demonstrated anti-leukemic activity by targeting leukemic stem cells (LSCs) with high aldehyde dehydrogenase (ALDH) activity. In this study, we demonstrate ISA induces apoptosis by downregulating antiapoptotic MCL1 levels in pediatric and adult AML cell lines. Based on the inhibitory effects of Isatin analogs on key signaling pathways (PI3K/AKT, STAT3) and MCL1, we combined ISA with a BCL2 inhibitor, VEN, for effective AML treatment. The combination of ISA with VEN exhibited synergetic cytotoxicity in naïve (MOLM-13, MV4-11) and VEN-resistant (HL-60, OCI-AML3, U937, and KG-1) human AML cell lines by bliss analysis. ISA also showed significant synergetic cytotoxicity in combination with ABT-737, a BH3 mimetic inhibitor of Bcl-xL, Bcl-2, and Bcl-w. Combinatorial treatment of AML cell lines with sub-lethal doses of ISA and VEN showed a significant increase in cellular apoptosis as indicated by the levels of annexin V measured by flow cytometry. Immunoblotting analysis after co-treatment with ISA and VEN showed downregulation of MCL1 levels along with alteration of other key apoptosis regulators in both VEN-sensitive (MV4-11) and inherently VEN-resistant (U937) AML cell lines. Current and future studies will primarily focus on further exploiting the molecular mechanism of action for the combinatorial approach. Furthermore, our aim will be to demonstrate activity in primary patient cells along with preclinical efficacy in AML animal models. To summarize, in vitro findings showed synergetic cooperation between ISA and VEN in combination for effective induction of apoptosis and anti-leukemic activity in AML cells that are sensitive and resistant to VEN. Citation Format: Upendarrao Golla, Satyam Patel, Jeremy Hengst, Charyguly Annageldiyev, Joseph Schramm, Shantu Amin, Dhimant Desai, David Claxton, Sinisa Dovat, Arati Sharma. Isatin analogs potentiate the cytotoxicity of venetoclax in acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7227.

Abstract 3270: SH3GLB1 is involved with hydrogen peroxide/akt signaling in glioblastoma multiforme cells

Abstract Glioblastoma (GBM), a malignant brain tumor, has poor survival outcomes due to recurrence or drug resistance. In our previous studies, it was found that superoxide dismutase 2 (SOD2) was associated with temozolomide (TMZ) resistance. Moreover, SOD2 can convert O2 •− to H2O2, and H2O2 redox signaling is vital for physiological reactions and promotes tumor progression. We used cell lines, patient primary cells and pharmacological inhibitors/activators to confirm the significance of H2O2 signaling. We used the cultured cells to verify roles of H2O2 signaling in the plasticity of cell fate and did animal experiments to identify the optimal treatment strategy. In the present study, SOD2 was found to be the upstream mediator, and combining SOD inhibitor and TMZ, the cells showed reduced SH3GLB1 and autophagy levels. We then found that SH3GLB1 was regulated by H2O2 through AKT signaling using experiments of regulating redox homeostasis. The change of intracellular H2O2 levels in mitochondria following the treatments is similar to that in cytosol, however, the cell fate of parental or resistant cells was quite diverse. Notably, CCCP, a mitochondrial membrane potential disrupter, increased endogenous H2O2 production, and TMZ-increased SH3GLB1 levels can be reversed by HgCl2, reflecting the aquaporin function. In animal models, a combination of TMZ and 4-hydroxynonenal reduced growth of the SH3GLB1-knockdown resistant cells, along with suppressed autophagy levels. Our results characterizing the mechanism of H2O2-SH3GLB1 axis signaling in acquired TMZ resistance of GBM cells will provide new therapeutic strategies against the disease in the future. Citation Format: Chia-Hung Chien, Kwang-Yu Chang. SH3GLB1 is involved with hydrogen peroxide/akt signaling in glioblastoma multiforme cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3270.

Comprehensive characterization of IFNγ signaling in acute myeloid leukemia reveals prognostic and therapeutic strategies

Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation, inflammation, and tumor surveillance. However, while IFNγ levels were elevated in sera of most newly diagnosed acute myeloid leukemia (AML) patients, its complex interplay in AML remains insufficiently understood. We aim to characterize these complex interactions through comprehensive bulk and single-cell approaches in bone marrow of newly diagnosed AML patients. We identify monocytic AML as having a unique microenvironment characterized by IFNγ producing T and NK cells, high IFNγ signaling, and immunosuppressive features. IFNγ signaling score strongly correlates with venetoclax resistance in primary AML patient cells. Additionally, IFNγ treatment of primary AML patient cells increased venetoclax resistance. Lastly, a parsimonious 47-gene IFNγ score demonstrates robust prognostic value. In summary, our findings suggest that inhibiting IFNγ is a potential treatment strategy to overcoming venetoclax resistance and immune evasion in AML patients.

Activation of Epstein-Barr Virus' Lytic Cycle in Nasopharyngeal Carcinoma Cells by NEO212, a Conjugate of Perillyl Alcohol and Temozolomide.

The Epstein-Barr virus (EBV) is accepted as a primary risk factor for certain nasopharyngeal carcinoma (NPC) subtypes, where the virus persists in a latent stage which is thought to contribute to tumorigenesis. Current treatments are sub-optimal, and recurrence occurs in many cases. An alternative therapeutic concept is aimed at triggering the lytic cycle of EBV selectively in tumor cells as a means to add clinical benefit. While compounds able to stimulate the lytic cascade have been identified, their clinical application so far has been limited. We are developing a novel anticancer molecule, NEO212, that was generated by covalent conjugation of the alkylating agent temozolomide (TMZ) to the naturally occurring monoterpene perillyl alcohol (POH). In the current study, we investigated its potential to trigger the lytic cycle of EBV in NPC cells in vitro and in vivo. We used the established C666.1 cell line and primary patient cells derived from the brain metastasis of a patient with NPC, both of which harbored latent EBV. Upon treatment with NEO212, there was an increase in EBV proteins Zta and Ea-D, key markers of the lytic cycle, along with increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP), a marker of endoplasmic reticulum (ER) stress, followed by the activation of caspases. These effects could also be confirmed in tumor tissue from mice implanted with C666.1 cells. Towards a mechanistic understanding of these events, we used siRNA-mediated knockdown of CHOP and inclusion of anti-oxidant compounds. Both approaches blocked lytic cycle induction by NEO212. Therefore, we established a sequence of events, where NEO212 caused reactive oxygen species (ROS) production, which triggered ER stress and elevated the levels of CHOP, which was required to stimulate the lytic cascade of EBV. Inclusion of the antiviral agent ganciclovir synergistically enhanced the cytotoxic impact of NEO212, pointing to a potential combination treatment for EBV-positive cancers which should be explored further. Overall, our study establishes NEO212 as a novel agent able to stimulate EBV's lytic cycle in NPC tumors, with implications for other virus-associated cancers.

Plasma-derived exosomal miR-326, a prognostic biomarker and novel candidate for treatment of drug resistant pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a cancer with high incidence rate in pediatrics and drug resistance is a major clinical concern for ALL treatment. The current study was designed to evaluate the role of exosomal miR-326 in diagnosis and treatment of children with B-ALL. Exosomes were isolated from plasma samples of 30 patients and B-ALL cell lines followed by characterization, using nanoparticle tracking analysis, immunoblotting assay and electron microscopy. qPCR showed significantly increased levels of miR-326 in patients exosomes compared with non-cancer controls (P < 0.05, AUC = 0.7500). Moreover, a comparison between the sensitive and drug resistant patients revealed a prognostic value for the exosomal miR326 (P < 0.05, AUC = 0.7755). Co-culture studies on drug resistant patient primary cells and B-ALL cell lines suggested that exosomes with high miR-326 level act as vehicles for reducing cells viability. B-ALL cell line transfection with naked miR-326 mimic confirmed the results, and fluorescence microscopy validated uptake and internalization of exosomes by target cells. The novel introduced features of the exosomal miR-326 address a non-invasive way of diagnosing primary drug resistance in pediatric ALL and advocates a novel therapeutic strategy for this cancer.

The Development of an Advanced Model for Multilayer Human Skin Reconstruction In Vivo.

Human skin reconstruction on immune-deficient mice has become indispensable for in vivo studies performed in basic research and translational laboratories. Further advancements in making sustainable, prolonged skin equivalents to study new therapeutic interventions rely on reproducible models utilizing patient-derived cells and natural three-dimensional culture conditions mimicking the structure of living skin. Here, we present a novel step-by-step protocol for grafting human skin cells onto immunocompromised mice that requires low starting cell numbers, which is essential when primary patient cells are limited for modeling skin conditions. The core elements of our method are the sequential transplantation of fibroblasts followed by keratinocytes seeded into a fibrin-based hydrogel in a silicone chamber. We optimized the fibrin gel formulation, timing for gel polymerization in vivo, cell culture conditions, and seeding density to make a robust and efficient grafting protocol. Using this approach, we can successfully engraft as few as 1.0 × 106 fresh and 2.0 × 106 frozen-then-thawed keratinocytes per 1.4 cm2 of the wound area. Additionally, it was concluded that a successful layer-by-layer engraftment of skin cells in vivo could be obtained without labor-intensive and costly methodologies such as bioprinting or engineering complex skin equivalents. Key features • Expands upon the conventional skin chamber assay method (Wang et al., 2000) to generate high-quality skin grafts using a minimal number of cultured skin cells. • The proposed approach allows the use of frozen-then-thawed keratinocytes and fibroblasts in surgical procedures. • This system holds promise for evaluating the functionality of skin cells derived from induced pluripotent stem cells and replicating various skin phenotypes. • The entire process, from thawing skin cells to establishing the graft, requires 54 days. Graphical overview.

Role of mitochondrial fusion proteins MFN2 and OPA1 on lung cellular senescence in chronic obstructive pulmonary disease

BackgroundMitochondrial dysfunction and lung cellular senescence are significant features involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) stands as the primary contributing factor to COPD. This study examined mitochondrial dynamics, mitophagy and lung cellular senescence in COPD patients and investigated the effects of modulation of mitochondrial fusion [mitofusin2 (MFN2) and Optic atrophy 1 (OPA1)] on CS extract (CSE)-induced lung cellular senescence.MethodsSenescence-associated secretory phenotype (SASP) component mRNAs (IL-1β, IL-6, CXCL1 and CXCL8), mitochondrial morphology, mitophagy and mitochondria-related proteins (including phosphorylated-DRP1(p-DRP1), DRP1, MFF, MNF2, OPA1, PINK1, PARK2, SQSTM1/p62 and LC3b) and senescence-related proteins (including P16, H2A.X and Klotho) were measured in lung tissues or primary alveolar type II (ATII) cells of non-smokers, smokers and COPD patients. Alveolar epithelial (A549) cells were exposed to CSE with either pharmacologic inducer (leflunomide and BGP15) or genetic induction of MFN2 and OPA1 respectively.ResultsThere were increases in mitochondrial number, and decreases in mitochondrial size and activity in lung tissues from COPD patients. SASP-related mRNAs, DRP1 phosphorylation, DRP1, MFF, PARK2, SQSTM1/p62, LC3B II/LC3B I, P16 and H2A.X protein levels were increased, while MFN2, OPA1, PINK1 and Klotho protein levels were decreased in lung tissues from COPD patients. Some similar results were identified in primary ATII cells of COPD patients. CSE induced increases in oxidative stress, SASP-related mRNAs, mitochondrial damage and dysfunction, mitophagy and cellular senescence in A549 cells, which were ameliorated by both pharmacological inducers and genetic overexpression of MFN2 and OPA1.ConclusionsImpaired mitochondrial fusion, enhanced mitophagy and lung cellular senescence are observed in the lung of COPD patients. Up-regulation of MFN2 and OPA1 attenuates oxidative stress, mitophagy and lung cellular senescence, offering potential innovative therapeutic targets for COPD therapy.Graphical

Long non-coding RNA growth arrest-specific 5 inhibits liver fibrogenesis in biliary atresia by interacting with microRNA-222 and repressing IGF1/AKT signaling.

Long non-coding RNA growth arrest-specific 5 (lncRNA GAS5) has been shown to inhibit liver fibrosis through serving as a competing endogenous RNA for microRNA-222 (miR-222). Progressive liver fibrosis is a typical characteristic of biliary atresia (BA). However, the role of GAS5/miR-222 and its underlying mechanisms remain largely unknown in BA. The expression of GAS5 was determined in the liver and primary hepatic stellate cells (HSCs) of BA patients. Then, the effects of GAS5 on the activation and proliferation of HSCs were evaluated. Furthermore, the interaction between GAS5 and miR-222 was investigated by a luciferase gene report assay. Next, the effects of IGF1/AKT signaling were determined to clarify the downstream mechanism of GAS5. Finally, GAS5 administration was performed to explore its role in an experimental BA mouse model. GAS5 expression was decreased in liver tissues and HSCs of BA patients, and was inversely correlated with liver fibrosis in BA. Up-regulation of GAS5 in LX-2 cells significantly reduced smooth muscle α-actin (α-SMA) and collagen 1a1 (COL1A1) expression, inhibited cell proliferation and clone formation ability, induced S phase increase, and promoted cell apoptosis. Moreover, GAS5 was negatively regulated by miR-222, which promoted HSCs activation and proliferation, and was positively correlated with liver fibrosis in BA. Additionally, the expressions of IGF1, p-PI3K, and p-AKT were decreased when LX-2 cells over-expressed GAS5, whereas knockdown of IGF1 or AKT significantly decreased α-SMA and COL1A1 expression, suppressed cell proliferation, and enhanced cell apoptosis in LX-2 cells. Furthermore, GAS5 administration significantly increased apoptosis and reduced liver fibrosis, α-SMA and COL1A1 expressions in liver tissues of BA mice. GAS5 inhibited liver fibrosis in BA by interacting with miR-222 and regulating IGF1/AKT signaling, which may be a therapeutic target to alleviate liver fibrosis in BA.

Restoration of Mir-185 in Combination with BCR-ABL Downregulation By Therapeutic Delivery of siRNA with Lipid Nanoparticle Carriers Targets Drug Resistant Leukemic Stem/Progenitor Cells

Background: Despite development of effective tyrosine kinase inhibitors (TKIs) for chronic myeloid leukemia (CML), TKI therapy is not curative as some patients develop drug resistance or are at risk of disease relapse. Importantly, CML leukemic stem cells (LSCs) remain largely unresponsive and represent a key population for sustaining disease and relapse. We previously demonstrated that miR-185 levels were significantly reduced in CD34 + stem/progenitor cells from TKI-nonresponders compared to TKI responders. Further, restoration of miR-185 levels in CML LSCs and their progenitors impaired their survival and sensitized them to TKI in vitro and in vivo. However, the therapeutic potential of downregulating BCR-ABL using siRNA in combination with miR-185 mimic treatment to target CML LSCs have yet to be elucidated. Objective: Downregulation of BCR-ABL using siRNA represents a promising therapeutic approach to inhibit its kinase and non-kinase functions to combat drug-resistant CML and overcome limitations of current therapy. Additionally, previous work suggests that restoring miR-185 levels may improve therapeutic outcomes from complimentary therapies in CML, particularly in combination with BCR-ABL inhibition. Therefore, we aim to investigate the therapeutic outcomes and mechanism of action of inhibiting BCR-ABL using siRNA in combination with miR-185 mimic treatment in drug-resistant CML. Methods: To assess the effects of BCR-ABL downregulation in combination with miR-185 restoration, we encapsulated siRNA targeting BCR-ABL and a miR-185 mimic using an optimized lipid nanoparticle (LNP) formulation. Biological effects and mechanistic changes of this novel RNA combination was evaluated using various CML cell lines expressing b2a2, b3a2, T315I mutated BCR-ABL and CD34 + stem/progenitor cells from non-responder patients. Results: A total of 7 siRNA sequences targeting various fusion regions of BCR-ABL were designed and tested to effectively target primitive CML cells. The highest performing BCR-ABL siRNA encapsulated showed significant knockdown in various CML models expressing b2a2, b3a2 and T315I mutated BCR-ABL (65-99% knockdown). After 48 hours of 0.5 μg/mL BCR-ABL siRNA LNP treatment, BCR-ABL protein levels and its direct downstream target p-CrkL are undetectable by immunoblotting. The robust downregulation of BCR-ABL results in a dose-dependent decrease in cell viability (down to 28% and 9.4% viable cells at a dose of 1 μg/mL) and increase in apoptotic cells (70% and 78% Annexin-V positive) in imatinib resistant K562 and T315I mutated cells, respectively after 72 hours. Similarly, encapsulated miR-185 mimic downregulates its main downstream target PAK6 transcript levels down to 50% in drug sensitive and drug-resistant cell lines and in TKI nonresponder CD34 + stem/progenitor cells. More importantly, our RNA LNP formulation demonstrated significant uptake (&amp;gt; 80% Cy5 positive) of fluorescent-labelled siRNA in TKI nonresponder CD34 + stem/progenitor cells and decreased BCR-ABL mRNA transcript levels by 60%. The combined BCR-ABL inhibition and miR-185 mimic treatment were determined to be highly synergistic when analysed using the Chou-Talalay (combination index less than 1), ZIP, Bliss and HSA methods (synergy scores larger than 10) in both drug-sensitive and drug-resistant CML cells. Furthermore, treatment of BCR-ABL siRNA and miR-185 using LNPs decreased the colony forming units of nonresponder patient cells by 50%. Interestingly, this effect was further enhanced when combined with TKIs, decreasing colony forming units to 20% compared to control after 14 days. Conclusion: We have uncovered a LNP formulation that is able to effectively deliver therapeutic BCR-ABL siRNA and miR-185 mimic into primitive CML cells. This combination treatment significantly reduces the levels of oncogenic BCR-ABL and PAK6, decreasing the viability clonogenic potential and increases apoptosis in CML cell line models and primary patient cells. Importantly, this combination strategy overcomes TKI resistance in CD34 + stem/progenitor cells suggesting a feasible and effective model for patients suffering from clinical resistance and relapse.

Inactivation of DNA Polymerase Theta (PolΘ) Is Synthetic Lethal in DNMT3A Mutated Myeloid Malignancies - Potential Clinical Applications

Somatic mutations in DNMT3A are associated with unfavorable outcome in patients with AML, MPN and CML. DNMT3A mutations promote resistance to anthracyclines (including daunorubicin, the component of standard “7+3” induction therapy), interferon alpha, and ABL1 kinase inhibitor imatinib. Thus, malignant clones carrying DNMT3A mutations may be difficult to eliminate using standard treatments. AML, MPN and CML cells harbor oncogenic tyrosine kinase (OTK) such as FLT3(ITD), JAK2(V617F) and BCR-ABL1, respectively. We reported before that elevated levels of formaldehyde generated by altered serine/one-carbon cycle metabolism contributed to accumulation of highly lethal DNA double-strand breaks (DSBs) in OTK-positive cells. To protect malignant cells from DSB-induced apoptosis, OTKs regulate DNA damage response (DDR) mechanisms involving DSBs sensing (ATM and ATR kinases) and repairing (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, Polθ-dependent microhomology-mediated end-joining = TMEJ) as well as these activating cell cycle checkpoints (CHK1 and CHK2 kinases). Unfortunately, DNMT3A mutations caused resistance of OTK-positive cells to numerous DDR inhibitors (DDRis). DDRis sensitivity screen and synthetic lethal CRISPR/Cas9 screen revealed that OTK-positive cells with DNMT3A mutations are uniquely sensitive to the inhibition of DNA polymerase theta (Polθ encoded by POLQ gene). This effect was dependent on generation of formaldehyde by serine/one-carbon cycle metabolism. Abrogation of DNMT3A function by CRISPR/Cas9 targeting, Cre-loxP gene deletion, and heterozygous R882H mutation resulted in hypersensitivity of OTK-positive murine AML-like cells and human primary AML cells to Polθ inhibitors (Polθis) due to accumulation of toxic DSBs and activation of cGAS/STING pro-apoptotic pathway. Moreover, simultaneous loss of functional DNMT3A combined with inactivation of Polθ (by CRISPR/Cas9 targeting, insertion of neo-resistance gene, and D2230A+Y2231A polymerase inactive mutant) caused accumulation of DSBs, and reduced OTK-driven clonogenic potential and leukemogenic activity in mice. Polθ is abundantly overexpressed in OTK-positive DNMT3A-deficient cells due to enhanced POLQ mRNA stability and elevated translation of Polθ protein but not due to altered POLQ methylation and Polθ protein stability. Polθ is a key element not only in TMEJ of DSBs with limited end-resection, but also in replication fork restart and in single-strand DNA (ssDNA) gap filling. OTK-positive DNMT3A-deficient cells displayed hyperactivity of Polθ-mediated TMEJ and replication fork restart, but not ssDNA gap filling. These effects were accompanied by increased loading of Polθ on DNA damage detected by Polθ foci formation and chromatin extraction. Moreover, DNMT3A deficiency modulates chromatin architecture at DSBs to limit DNA end-resection thus favoring TMEJ over HR. Furthermore, we tested the effectiveness of Polθis combined with FDA approved drugs (quizartinib, etoposide, cytarabine, azacytidine) against FLT3(ITD)-positive DNMT3A-deficient cells (primary patient cells and cell lines) in vitro and in vivo. The combination of Polθis + quizartinib and Polθis + etoposide completely eradicated clonogenic activity of these cells while Polθis + cytarabine and Polθis + azacytidine exerted modest and weak effects, respectively, when compared to individual compound treatments. These drug combinations were only modestly toxic to normal bone marrow cells. Treatment with Polθi or etoposide reduced the percentage of GFP+ FLT3(ITD)-positive DNMT3A-deficient leukemia cells in peripheral blood of the mice by ~2-fold and prolonged survival time by ~1.5-fold. Remarkably, the combination of Polθi and etoposide eradicated leukemia cells below detectable levels in 6/12 mice with no visible toxicity. Median survival time of the mice will be recorded. Altogether, we discovered that Polθ protects OTK-positive DNMT3A-deficient myeloid malignant cells from the toxic effects of DSBs and identified Polθ as a novel therapeutic target.