FLT3 inhibitor efficacy in AML with FLT3-ITD is short-lived, frequently due to new mutations, most commonly in NRAS. Sphingosine kinase 1 (SPHK1), which phosphorylates sphingosine to generate sphingosine-1-phosphate (S1P), is upregulated and localized to the plasma membrane in RAS-mutated cells. We studied S1P and FLT3 co-targeting to overcome FLT3 inhibitor resistance in NRAS-mutated FLT3-ITD AML cells. NRAS-mutated FLT3-ITD AML cell lines and patient blasts were treated with FLT3 inhibitors and/or S1P receptor (S1PR) modulators. FLT3 inhibitor sensitivity was assessed by immunoblotting, cytotoxicity, apoptosis and colony formation. Co-treatment was also assessed in vivo in an orthotopic mouse model. Downstream RAS and SPHK1 effectors were measured by immunoblotting and qRT-PCR. The S1PR modulators fingolimod (FTY720) and mocravimod (KRP-203) resensitized FLT3-ITD-expressing MOLM-14 and MV4-11 human AML cells with G12D, G12S, Q61K or Q61H, but not G12C, and patient blasts with G13D, G13V or G12D NRAS mutations to FLT3 inhibitors. Moreover, FTY720 co-treatment resensitized G12D NRAS-mutated M14(R)701 cells to gilteritinib in vivo. Co-treatment inactivated ERK, transcriptionally downregulated SPHK1, and inactivated downstream AKT, p70 S6K and BAD, with inactivation abrogated by constitutive SPHK1 expression. The clinically applicable S1PR modulators fingolimod and mocravimod resensitize NRAS-mutated FLT3-ITD AML cells to FLT3 inhibitors, supporting potential clinical efficacy.

FLT3 inhibitors have improved outcomes in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), but responses are not durable. Notably, FLT3 inhibitors clear blasts from the blood, but not the bone marrow, a hypoxic niche. We investigated effects of hypoxia and the key nutrient glutamine on FLT3 inhibitor response. FLT3-ITD AML cell lines and patient blasts were cultured with FLT3 inhibitors under normoxia (21%) or hypoxia (<1% O₂) with or without glutamine or the glutaminase inhibitor telaglenastat (CB-839). Cytotoxicity was measured in WST-1 assays and drug combination effects by Chou-Talalay analysis. Protein expression was measured by immunoblotting, turnover and proteasomal degradation by cycloheximide chase with and without MG-132, and mRNA expression by RT-qPCR. Effect of the ubiquitin ligase c-CBL was tested by siRNA knockdown. FLT3 inhibitor IC₅₀s were 3-5-fold higher in hypoxia than normoxia, associated with FLT3-ITD and p-STAT5 downregulation and accelerated FLT3-ITD proteasomal degradation (half-life, 1.0 vs. 2.5 hours). c-CBL expression increased in hypoxia, and c-CBL knockdown restored FLT3-ITD expression and FLT3 inhibitor sensitivity. Glutamine deprivation or telaglenastat treatment abrogated c-CBL upregulation in hypoxia and preserved FLT3-ITD and p-STAT5 expression and FLT3 inhibitor sensitivity. Telaglenastat synergized with FLT3 inhibitors in hypoxia, supporting clinical testing.

Artificial intelligence (AI) has become an indispensable ally in virology, enabling the analysis of enormous datasets that extend from viral genomes to behavioral and clinical information. HIV-1, a rapidly evolving retrovirus with extraordinary genetic diversity and a persistent latent reservoir, poses unique computational challenges that are now approachable through data-driven models. Modern machine-learning and deep-learning architectures can decode viral sequences, predict drug resistance and co-receptor usage, simulate evolutionary trajectories under therapy, and integrate multi-omics information to identify molecular determinants of persistence. In parallel, AI-assisted chemoinformatic shortens drug-discovery cycles, while network and language models enhance epidemiological surveillance and individualized care. The convergence of AI with organoid technologies, single-cell systems biology, and population informatics is redefining HIV research from static observation to dynamic prediction. Ethical transparency, algorithmic fairness, and equitable access remain central to ensuring that these innovations accelerate-not distort-the path toward durable remission and cure.

Hematological malignancies such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphomas, and multiple myeloma remain difficult to model ex vivo because conventional two-dimensional (2D) cultures and murine systems fail to reproduce the spatial, metabolic, vascular, and immune complexity of human bone marrow and lymphoid niches. Recent advances in three-dimensional (3D) platforms-including spheroids, engineered organoid-like marrow models, and microfluidic niche-on-a-chip systems-now allow for a more physiological replication of stromal, endothelial, and immune interactions that drive resistance and relapse. In this review, we introduce explicit definitions distinguishing spheroids, organoid-like constructs, true hematopoietic organoids, and microfluidic devices to establish a unified framework for hematologic 3D modeling. We synthesize applications across AML, CML, lymphoma, and myeloma, highlighting mechanistic insights, strengths, and limitations unique to each disease. Finally, we outline a translational roadmap that integrates bioprinting, perfusable vasculature, immune reconstitution, and AI-driven analytics toward next-generation patient-specific platforms. These innovations position 3D marrow-mimetic systems as essential tools for precision oncology in blood cancers.

FLT3-ITD is present in acute myeloid leukemia (AML) in 25% of patients and is associated with poor treatment outcomes. FLT3 inhibitors effectively target FLT3-ITD, but resistance develops during FLT3 inhibitor treatment, frequently due to new mutations that activate the Ras-Raf-MEK-ERK signaling pathway. NRAS mutations altering RAS protein amino acids at codons G12, G13 or Q61 constitutively activate RAS proteins by impairing GTPase activity, with consequent constitutive activation of the Ras-Raf-MEK-ERK pathway, are most commonly associated with development of resistance to FLT3 inhibitors during treatment. Sphingosine kinase 1 (SphK1) is upregulated and localized to the plasma membrane in RAS-mutated cells. SphK1 phosphorylates sphingosine to form the pro-survival lipid sphingosine-1-phosphate (S1P), which promotes cell survival and proliferation and inhibits apoptosis. SphK1 is linked to FLT3 inhibitor resistance, as prolonged sorafenib exposure was shown to activate the Sphk1/S1P axis. Here we studied the efficacy of targeting Sphk1 with sphingosine-1-phosphate receptor (S1PR) modulators in conjunction with FLT3 inhibitors to overcome FLT3 inhibitor resistance mediated by NRAS mutations in AML cells with FLT3-ITD. Methods MOLM-14 and MV4-11 human FLT3-ITD AML cell lines with NRAS mutations including G12D, G12S, G12C, Q61K and Q61H and FLT3-ITD AML patient blasts with G13V and G13D mutations were cultured with the FLT3 inhibitors gilteritinib (10 nM) or quizartinib (1 nM) and/or the S1PR modulators fingolimod (FTY720; 2.5 μM) or mocravimod (KRP203; 5 μM). Apoptosis was measured by Annexin V and propidium iodide labeling, detected by flow cytometry. p-FLT3 (Y591), FLT3, p-STAT5 (Y694), STAT5, Sphk1, p-AKT (S473), AKT, p-p70 S6K (T389), p70 S6K, p-BAD (S136) and BAD protein expression were measured by immunoblotting, and Sphk1 mRNA by qPCR. MOLM-14 cells with an NRAS G12D mutation infected with lentiviral pLenti-Sphk1 plasmid overexpressing Sphk1 and pLenti-empty vector control were treated with gilteritinib and FTY720 combination to confirm the key role of Sphk1 in FLT3 inhibitor resensitization and inactivation of Sphk1 downstream proteins. To test efficacy in vivo, NSG mice engrafted with NRAS G12D-mutated MOLM-14 cells following intravenous injection were treated with the FLT3 inhibitor gilteritinib (7.5 mg/kg) and/or the S1PR modulator FTY720 (10 mg/kg), or vehicle control. Leukemia burden was assessed weekly by non-invasive luciferin imaging and compared by 2-Way ANOVA with Sidak's multiple comparison test. Survival was compared by Kaplan-Meier analysis. Results The S1PR modulators fingolimod (FTY720) and mocravimod (KRP203) resensitized FLT3-ITD-expressing MOLM-14 and MV4-11 cells with NRAS G12D, G12S, Q61K or Q61H, but not G12C, mutations to the FLT3 inhibitors gilteritinib and quizartinib, evidenced by decreased FLT3 and STAT5 phosphorylation and increase in apoptotic cells from 5% up to 90%. Concurrent treatment of G12D, G12S, Q61K and Q61H, but not G12C, NRAS-mutated MOLM-14 and MV4-11 cells with S1PR modulators and FLT3 inhibitors transcriptionally downregulated Sphk1 and inactivated downstream AKT, p70S6K and BAD. Constitutive Sphk1 expression in G12D NRAS-mutated MOLM-14 cells abrogated AKT, p70S6K and BAD inactivation and FLT3 inhibitor resensitization by gilteritinib and FTY720 combination treatment. FTY720 co-treatment overcame gilteritinib resistance of G12D NRAS-mutated MOLM-14 cells in vivo, evidenced by decreased luminescence (p=0.015) and prolonged survival (p=0.0035), comparing gilteritinib and FTY720 to single-agent gilteritinib. Conclusions The S1PR agonists fingolimod (FTY720) and mocravimod (KRP203) resensitize FLT3-ITD AML cells harboring G12D, G12S, Q61K, and Q61H, but not G12C, NRAS mutations to FLT3 inhibitors. The data support potential clinical efficacy of combination regimens with these clinically applicable drugs to overcome FLT3 inhibitor resistance driven by most NRAS mutations.

Exaggerated inflammation and cytokine storm are hallmark features of influenza A virus (IAV)-induced respiratory diseases. While previous studies unequivocally demonstrated the pathophysiological consequences (multiorgan failure) of IAV-associated cytokine storm, it remains unknown if IAV-induced systemic inflammation impacts the fitness and differentiation of immune cells from hematopoietic stem cells (HSCs). Our data on lethal IAV-infected C57BL/6 wildtype mice after 10 days of infection indicated reduced monocyte- and lymphocyte- counts in the peripheral blood, and overall cellularity of spleen, thymus and lymph nodes. IAV- infection resulted in increased numbers of myeloid cells, CD8+ T cells, alveolar macrophages (AVMs), CD11b+ dendritic cells (DCs) & plasmacytoid DCs (pDCs), whereas decreased frequencies of CD103+ DCs, in the lungs of IAV-infected mice. Analysis of spleen and draining lymph nodes indicated reduced absolute numbers of B cells, T cells, monocytes and DCs after 10 days of lethal IAV infection. Thymic analysis indicated perturbed T cell differentiation and bone marrow (BM) data revealed impaired DC differentiation following IAV infection. Hematopoietic stem and progenitor cells (HSPCs) studies demonstrated an imbalanced distribution of HSCs, multipotent progenitors (MPPs), myeloid progenitors and DC progenitors within the BM niche. Mechanistic studies exhibited elevated levels of systemic inflammation and altered local pro-inflammatory milieu. Molecular analyses documented elevated levels of intracellular reactive oxygen species (ROS) at all stages of HSPC differentiation and increased mass of active mitochondria in HSPC subsets. In essence, our studies provide novel insights into mechanisms through which lethal IAV-infection induces deficiencies of the innate and adaptive immune system.

This review underscores the important role of nutrition in enhancing the management of Human Immunodeficiency Virus type 1 (HIV-1). Highlighting the efficacy of dietary interventions, including, the importance of omega-3 fatty acids, vitamins D and B-12, and the Mediterranean diet, we delineate how these beneficial nutritional strategies can improve the effectiveness of combined antiretroviral therapy (cART), mitigate its side effects, and ameliorate metabolic disorders in people living with HIV-1 (PLWH). Our review advocates for the integration and implementation of personalized nutritional assessments into the care plan for PLWH, proposing actionable strategies for healthcare providers in HIV-1 field. Summarizing the current standing of the relevance of the nutritional and well-planned diet recommended for the PLWH and emphasizing on the future research directions, this review establishes a foundation for nutrition as a cornerstone in comprehensive HIV-1 management. Our review aims to improve patients’ health outcomes and overall quality of life for PLWH.

Pim inhibitor and gilteritinib combination treatment is synergistic in cells with FLT3-ITD. A,C, Apoptosis induction. Ba/F3-ITD, MV4-11, and MOLM-14 cells were treated with the FLT3 inhibitor gilteritinib (15 nmol/L for Ba/F3-ITD and 10 nmol/L for MV4-11 and MOLM-14) and/or the Pim inhibitor AZD1208 (1 µmol/L; A) or TP-3654 (1 µmol/L; C) or DMSO control for 48 hours in triplicate experiments. Apoptosis was analyzed by Annexin V and PI staining, measured by flow cytometry. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01. B,D, Cytotoxicity. Ba/F3-ITD cells seeded at 5,000 cells/well and MV4-11 and MOLM-14 cells at 10,000 cells/well in 96-well plates were treated for 48 hours with gilteritinib and/or AZD1208 (B) or TP-3654 (D) as single drugs and in combinations at the concentrations shown, in triplicate. Cytotoxicity was measured by the WST-1 assay, and drug combination effects were determined by Chou-Talalay analysis. Synergism was defined by combination index values < 0.8.

Background Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by cancer-associated bacteria (CAB) that impair tumor suppressor functions. Our previous research found that Mycoplasma fermentans DnaK, a chaperone protein, impairs p53 activities, which are essential for most anti-cancer chemotherapeutic responses. Methods To investigate the role of DnaK in chemotherapy, we treated cancer cell lines with M. fermentans DnaK and then with commonly used p53-dependent anti-cancer drugs (cisplatin and 5FU). We evaluated the cells’ survival in the presence or absence of a DnaK-binding peptide (ARV-1502). We also validated our findings using primary tumor cells from a novel DnaK knock-in mouse model. To provide a broader context for the clinical significance of these findings, we investigated human primary cancer sequencing datasets from The Cancer Genome Atlas (TCGA). We identified F. nucleatum as a CAB carrying DnaK with an amino acid composition highly similar to M. fermentans DnaK. Therefore, we investigated the effect of F. nucleatum DnaK on the anti-cancer activity of cisplatin and 5FU. Results Our results show that both M. fermentans and F. nucleatum DnaKs reduce the effectiveness of cisplatin and 5FU. However, the use of ARV-1502 effectively restored the drugs' anti-cancer efficacy. Conclusions Our findings offer a practical framework for designing and implementing novel personalized anti-cancer strategies by targeting specific bacterial DnaKs in patients with poor response to chemotherapy, underscoring the potential for microbiome-based personalized cancer therapies.

Abstract Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) has poor outcomes. FLT3-ITD drives constitutive and aberrant FLT3 signaling, activating STAT5 and upregulating the downstream oncogenic serine/threonine kinase Pim-1. FLT3 inhibitors are in clinical use, but with limited and transient efficacy. We previously showed that concurrent treatment with Pim and FLT3 inhibitors increases apoptosis induction in FLT3-ITD–expressing cells through posttranslational downregulation of Mcl-1. Here we further elucidate the mechanism of action of this dual targeting strategy. Cytotoxicity, apoptosis and protein expression and turnover were measured in FLT3-ITD–expressing cell lines and AML patient blasts treated with the FLT3 inhibitor gilteritinib and/or the Pim inhibitors AZD1208 or TP-3654. Pim inhibitor and gilteritinib cotreatment increased apoptosis induction, produced synergistic cytotoxicity, downregulated c-Myc protein expression, earlier than Mcl-1, increased turnover of both proteins, which was rescued by proteasome inhibition, and increased efficacy and prolonged survival in an in vivo model. Gilteritinib and Pim inhibitor cotreatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids, preventing phosphorylation at these sites, did not downregulate these proteins, increase their turnover or increase apoptosis induction. Moreover, concurrent treatment with gilteritinib and Pim inhibitors dephosphorylated (activated) the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β), and GSK-3β inhibition prevented c-Myc and Mcl-1 downregulation and decreased apoptosis induction. The data are consistent with c-Myc T58 and Mcl-1 S159 phosphorylation by activated GSK-3β as the mechanism of action of gilteritinib and Pim inhibitor combination treatment, further supporting GSK-3β activation as a therapeutic strategy in FLT3-ITD AML. Significance: FLT3-ITD is present in 25% of in AML, with continued poor outcomes. Combining Pim kinase inhibitors with the FDA-approved FLT3 inhibitor gilteritinib increases cytotoxicity in vitro and in vivo through activation of GSK-3β, which phosphorylates and posttranslationally downregulates c-Myc and Mcl-1. The data support efficacy of GSK-3β activation in FLT3-ITD AML, and also support development of a clinical trial combining the Pim inhibitor TP-3654 with gilteritinib.

Well-controlled repair mechanisms are involved in the maintenance of genomic stability, and their failure can precipitate DNA abnormalities and elevate tumor risk. In addition, the tumor microenvironment, enriched with factors inducing oxidative stress and affecting cell cycle checkpoints, intensifies DNA damage when repair pathways falter. Recent research has unveiled associations between certain bacteria, including Mycoplasmas, and various cancers, and the causative mechanism(s) are under active investigation. We previously showed that Mycoplasma fermentans DnaK, an HSP70 family chaperone protein, hampers the activity of proteins like PARP1 and p53, crucial for genomic integrity. Moreover, our analysis of its interactome in human cancer cell lines revealed DnaK's engagement with several components of DNA-repair machinery. Finally, in vivo experiments performed in our laboratory using a DnaK knock-in mouse model generated by our group demonstrated that DnaK exposure led to increased DNA copy number variants, indicative of genomic instability. We present here evidence that expression of DnaK is linked to increased i) incidence of tumors in vivo upon exposure to urethane, a DNA damaging agent; ii) spontaneous DNA damage ex vivo; and iii) expression of proinflammatory cytokines ex vivo, variations in reactive oxygen species levels, and increased β-galactosidase activity across tissues. Moreover, DnaK was associated with increased centromeric instability. Overall, these findings highlight the significance of Mycoplasma DnaK in the etiology of cancer and other genetic disorders providing a promising target for prevention, diagnostics, and therapeutics.

Human immunodeficiency virus type 1 (HIV-1) continues to pose a significant global health challenge despite advances in combined antiretroviral therapy (cART), which has transformed HIV-1 infection from a fatal disease to a manageable chronic condition. However, cART is not curative, and its long-term use is associated with challenges such as pill burden, drug toxicities, and the emergence of drug-resistant viral strains. The persistence of active viral reservoirs necessitates lifelong treatment, highlighting the need for alternative therapeutic strategies capable of achieving HIV-1 remission or cure. Stem cell therapy has emerged as a promising approach to address these challenges by targeting latent viral reservoirs, restoring host immune function, and potentially achieving sustained viral suppression in the absence of cART. This review critically evaluates current scientific literature on stem cell therapies for HIV-1, focusing on three major approaches: 1) hematopoietic stem cell transplantation (HSCT), 2) gene therapy, and 3) cell-based immunotherapies. Each approach is examined in terms of its underlying mechanisms, clinical feasibility, recent advancements, and associated challenges. Furthermore, future research directions are discussed, emphasizing the optimization of the current treatment protocols, enhancement of safety and efficacy, and the importance of large-scale clinical trials with different cohorts (different HIV clades, different genders of participants, and pediatric HIV) to evaluate long-term outcomes that include effective and scalable HIV cure challenges. Collaborative efforts across multidisciplinary fields are needed to overcome existing barriers so to realize the full therapeutic potential of stem cell-based approaches for developing an effective and scalable remission or cure strategies.

Range values of controls for the indicated experiments

Figure S2. KEGG/GO analysis of MIR300 on PP2A-regulated signal transduction pathways. Cartoon shows the SET-dependent PP2A Inhibitory pathway in CML and the pleiotropic inhibitory effect of PP2A activation on validated and predicted MIR300 targets regulating G1/S cell cycle transition, Wnt-beta-catenin, TGFbeta, JAK-STAT, PI-3K-Akt, RAS-MAPK and Notch signaling pathways.

<p>Figure S1. MIR300 activity in quiescent leukemic stem and progenitor cells. CFC-replating assays shows effects of lentiviral-mediated ectopic MIR300 expression, 250 nM and 500 nM CpG-miR-300 on serial replating activity (2nd replating) of leukemic chronic and acute CML and normal UCB CD34+CD38- HSC-enriched cell fractions. Infection with lentiviral empty vector and treatment with CpG-anti-MIR300 and CpG-scramble served as controls.</p>

<p>Figure S4. MIR300 anti-proliferative activity accounts for BMM-induced LSC entry into quiescence. A, Structure of 14q32 DLK1-DIO3 genomic imprinted locus hosting the MEG3-regulated human MIR300. B, MIR300 levels in 5-Aza- or DMSO-treated (24h) Ph+ cells. C, Effect of hypoxia on proliferation of CFSE+CD34+ CML-BC cells. D, left: Effect of MSC (HS-5)-derived CM on LAMA-84 proliferation expressed as fold changes of CFSE mean of fluorescence intensity (MFI)+/-SEM; middle: pro-apoptotic effect of PAD (FTY720; 2.5uM) and DMSO (control) on HS-5-cultured LAMA-84 cells; right: Effect of MSC (HS-5)-derived CM BCR-ABL1 expression (anti-ABL1) and activity anti-PY), phospho-BCR-ABL1, JAK2 expression and activity JAK2 Y1007/1008, PP2A activity (pPP2AY307 inactive form) and GRB2 used as a control (blots are representative of three independent experiments). E, Levels of C/EBPbeta and GRB2 mRNA and protein in HS-5 cells exposed to hypoxia (48h; 1% O2). F, Effect of neutralizing TGFbeta antibody (anti-TGFb Ab; 48h, 1.25 μg/ml) on MIR300 levels in CD34+ CML-BC cells. G, Effect of ectopic C/EBPalpha (MigR1-deltauORF-C/EBPalpha-HA) and C/EBPbeta (MigR1-C/EBPB-ERTAM) on MIR300 levels in K562 cells. Immunoblot shows levels of C/EBPbeta and GRB2 in normoxic and hypoxic K562 cells.</p>

The human microbiota affects critical cellular functions, although the responsible mechanism(s) is still poorly understood. In this regard, we previously showed that Mycoplasma fermentans DnaK, an HSP70 chaperone protein, hampers the activity of important cellular proteins responsible for DNA integrity. Here, we describe a novel DnaK knock-in mouse model generated in our laboratory to study the effect of M. fermentans DnaK expression in vivo. By using an array-based comparative genomic hybridization assay, we demonstrate that exposure to DnaK was associated with a higher number of DNA copy number variants (CNVs) indicative of unbalanced chromosomal alterations, together with reduced fertility and a high rate of fetal abnormalities. Consistent with their implication in genetic disorders, one of these CNVs caused a homozygous Grid2 deletion, resulting in an aberrant ataxic phenotype that recapitulates the extensive biallelic deletion in the Grid2 gene classified in humans as autosomal recessive spinocerebellar ataxia 18. Our data highlight a connection between components of the human urogenital tract microbiota, namely Mycoplasmas, and genetic abnormalities in the form of DNA CNVs, with obvious relevant medical, diagnostic, and therapeutic implications.

<p>Figure S1. MIR300 activity in quiescent leukemic stem and progenitor cells. CFC-replating assays shows effects of lentiviral-mediated ectopic MIR300 expression, 250 nM and 500 nM CpG-miR-300 on serial replating activity (2nd replating) of leukemic chronic and acute CML and normal UCB CD34+CD38- HSC-enriched cell fractions. Infection with lentiviral empty vector and treatment with CpG-anti-MIR300 and CpG-scramble served as controls.</p>