Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myelodysplastic syndrome or myeloproliferative disorder for which hematopoietic stem cell transplantation remains the only curative option; however, outcomes are particularly poor in patients harboring PTPN11 (encodes SHP2 phosphatase) mutations. Using a Shp2E76K/+ JMML mouse model, we identify a pathogenic IL-17A/PTGS2/NLRP3 signaling axis that drives bone marrow inflammation, suppresses antitumor immunity, and promotes leukemic progression. Shp2E76K/+ mice exhibited profound immune dysregulation, characterized by expansion of regulatory T cells (Tregs), increased T-cell exhaustion, and impaired cytotoxic function with reduced CD4⁺ and CD8⁺ T-cell frequencies. Mechanistically, mutant macrophages upregulated IL-17A, triggering NLRP3 inflammasome activation, PTGS2 induction, caspase-1 cleavage, and IL-1β maturation, thereby amplifying inflammatory signaling within the marrow niche. Therapeutically, IL-17A neutralization suppressed inflammasome activity, while combined inhibition of NLRP3 and PTGS2 restored cytotoxic T-cell function, reduced systemic and marrow inflammation, reversed myeloproliferation, and significantly prolonged survival in Shp2E76K/+ mice. Importantly, ex vivo treatment of primary JMML patient samples with dual NLRP3/PTGS2 inhibition combined with MEK blockade significantly reduced leukemic progenitor colony formation, supporting translational relevance. In patient-derived xenograft models of PTPN11-mutant JMML, dual NLRP3/PTGS2 inhibition combined with MEK blockade most effectively reduced leukemic burden, decreased human CD45⁺ engraftment, and depleted leukemic CD34⁺CD38⁺ progenitors and GMPs while restoring MEP populations, resulting in significantly improved overall survival. Together, these findings establish IL-17A/PTGS2/NLRP3 signaling as a central driver of immune suppression and myeloid expansion in PTPN11-mutant JMML and highlight combinatorial anti-inflammatory targeting as a promising therapeutic strategy for this high-risk disease.

Stem cell biology has rapidly expanded into an interdisciplinary field with potential for next-generation cell-based therapies. However, a critical gap remains in our understanding of how physical forces influence stem cell behavior. Recent studies in mechanotransduction, the process by which cells sense and convert mechanical cues into biochemical signals, have revealed that biomechanical regulation is fundamental for stem cell fate, proliferation, and therapeutic efficacy. This review synthesizes recent findings on the intrinsic and extrinsic parameters of physical cues that govern mechanotransduction and shape stem cell biology, highlights the mechanosensitive responses, and explores how biomedical engineering (BME) can be employed to manipulate these processes to improve translational outcomes in clinical settings. By integrating insights from cell biology, mechanobiology, and engineering, this interdisciplinary field offers strategies to translate benchside discoveries into clinical applications, advancing the development of precise and effective stem cell-based therapies.

Abstract Introduction The cytoskeleton maintains cellular shape and structure, and serves as a biomechanical regulator of cellular responses. Although CD8+ T cell activation is well characterized, the roles of mechanotransduction and cytoskeletal stiffness remain unclear. Understanding how these biomechanical properties affect CD8+ T cell activation and function could provide new insight into immune regulation. Methods We examined CD8+ T cells from a Protein Tyrosine Phosphatase Non-Receptor Type 21 knockout (Ptpn21⁻/⁻, KO) mouse model, previously reported to exhibit a disorganized actin cytoskeleton in hematopoietic stem cells. Intrinsic biomechanical properties were quantified using atomic force microscopy (AFM) with Hertzian modeling. Functional consequences of altered stiffness were evaluated by flow cytometry, comparing KO and wild-type (WT) mice after in vitro stimulation and in vivo adoptive transfer of CD8+OT-I T cells into B16-OVA tumor-bearing recipients. Results AFM revealed that Ptpn21⁻/⁻ CD8+ T cells were mechanically softer with a reduced elastic modulus. This softening was functionally significant to CD8+ T cell behavior. KO cells showed reduced activation upon CD3/CD28 bead stimulation, while responses to soluble OVA peptide remained comparable, indicating defective mechanotransduction. Calcium-flux assays demonstrated reduced store-operated Ca²+ entry, and AFM showed prolonged relaxation time, together suggesting impaired mechanotransduction during early TCR signaling. In vivo, the percentage of KO CD8+ T cells circulating in the spleen, lymph nodes, and peripheral blood was significantly lower than that of WT cells in a competitive adoptive transfer model, and the residual KO cells were less activated and proliferative. Conclusion In conclusion, our findings demonstrate that cytoskeletal softening in Ptpn21⁻/⁻ CD8+ T cells impairs mechanotransduction, resulting in weakened T cell activation and reduced proliferation within a tumor-bearing environment. Funding Source HL162725, HL130995, CA282579 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Mutations in protein tyrosine phosphatase non-receptor type 11 (PTPN11) have been considered late acquired mutations in acute myeloid leukemia (AML) development. Using single-cell DNA sequencing, we found that PTPN11 mutations can occur as initiating events in some patients with AML when accompanied by strong oncogenic drivers, commonly NPM1 mutations. The resulting AML has a diverse set of variably differentiated myeloid cells with few myeloid cells that lack leukemic mutations. The role of Ptpn11 as a codriver was confirmed in a murine model that exhibits an AML phenotype with a comparable immune diversity that is serially engraftable and reconstituted from early precursor cells. Furthermore, lineage-negative bone marrow cells from these mice reconstitute the full diversity of mature myeloid cells, and these cells exhibit an altered cytokine response after physiologic stimulation. Our work highlights how PTPN11-mutated AML is derived from a multitude of codominant and late acquired aberrations that have a previously unrecognized differentiated myeloid clonal expansion potentially contributing to pathogenesis of the disease.

Chemotherapy is often a primary treatment for cancer. However, resistance leads to therapeutic failure. Acetylation dynamics play important regulatory roles in cancer cells, but the mechanisms by which acetylation mediates therapy resistance remain poorly understood. Here, using acetylome-focused RNA interference (RNAi) screening, we find that acetylation induced by mitochondrial dihydrolipoyl transacetylase (DLAT), independent of the pyruvate dehydrogenase complex, is pivotal in promoting resistance to chemotherapeutics, such as cisplatin. Mechanistically, DLAT acetylates methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) at lysine 44 and promotes 10-formyl-tetrahydrofolate (10-formyl-THF) and consequent mitochondrially encoded cytochrome c oxidase II (MT-CO2) induction. DLAT signaling is elevated in cancer patients refractory to chemotherapy or chemoimmunotherapy. A decoy peptide DMp39, designed to target DLAT signaling, effectively sensitizes cancer cells to cisplatin in patient-derived xenograft models. Collectively, our study reveals the crucial role of DLAT in shaping chemotherapy resistance, which involves an interplay between acetylation signaling and metabolic reprogramming, and offers a unique decoy peptide technology to overcome chemotherapy resistance.

Juvenile myelomonocytic leukemia (JMML) originates from mutated hematopoietic stem cells. The mechanism by which mutant stem cells are sustained, leading to leukemia development, remains elusive. By comprehensively examining transcriptomic profiles, cell compositions, developmental trajectories, and cell-cell interactions across various stages of tumor cell development in a mouse model of Ptpn11 mutation-associated JMML, we find that Ptpn11E76K/+ mutant stem cells exhibit de novo activation of the myeloid transcriptional program and markedly increased expression of innate immunity-associated antimicrobial peptides and pro-inflammatory proteins, particularly S100a9 and S100a8. Biological experiments confirm that S100a9/S100a8 confer a selective advantage to mutant stem cells through autocrine effects and facilitate immune evasion by recruiting and promoting immune-suppressive myeloid-derived suppressor cells in the microenvironment. Importantly, pharmacological inhibition of S100a9/S100a8 signaling effectively impede leukemia development from Ptpn11E76K/+ mutant stem cells. These findings collectively suggest that JMML-initiating cells exploit innate immune and inflammatory mechanisms to establish clonal dominance.

Copious expression of protein arginine methyltransferase 1 (PRMT1) is associated with poor survival in many types of cancers, including acute myeloid leukemia. We observed that a specific acute megakaryocytic leukemia (AMKL) cell line (6133) derived from RBM15-MKL1 knock-in mice exhibited heterogeneity in Prmt1 expression levels. Interestingly, only a subpopulation of 6133 cells expressing high levels of Prmt1 caused leukemia when transplanted into congenic mice. The PRMT1 inhibitor, MS023, effectively cured this PRMT1-driven leukemia. Seahorse analysis revealed that PRMT1 increased the extracellular acidification rate and decreased the oxygen consumption rate. Consistently, PRMT1 accelerated glucose consumption and led to the accumulation of lactic acid in the leukemia cells. The metabolomic analysis supported that PRMT1 stimulated the intracellular accumulation of lipids, which was further validated by fluorescence-activated cell sorting analysis with BODIPY 493/503. In line with fatty acid accumulation, PRMT1 downregulated the protein level of CPT1A, which is involved in the rate-limiting step of fatty acid oxidation. Furthermore, administering the glucose analog 2-deoxy-D-glucose delayed AMKL progression and promoted cell differentiation. Ectopic expression of Cpt1a rescued the proliferation of 6133 cells ectopically expressing PRMT1 in the glucose-minus medium. In conclusion, PRMT1 upregulates glycolysis and downregulates fatty acid oxidation to enhance the proliferation capability of AMKL cells.

The engraftment of haematopoietic stem and progenitor cells (HSPCs), particularly in cord-blood transplants, remains challenging. Here we report the role of the corticotropin-releasing hormone (CRH) in enhancing the homing and engraftment of human-cord-blood HSPCs in bone marrow through mechanical remodelling. By using microfluidics, intravital two-photon imaging and long-term-engraftment assays, we show that treatment with CRH substantially enhances HSPC adhesion, motility and mechanical remodelling, ultimately leading to improved bone-marrow homing and engraftment in immunodeficient mice. CRH induces Ras homologue gene family member A (RhoA)-dependent nuclear translocation of the yes-associated protein (YAP), which upregulates the expression of genes encoding extracellular-matrix proteins (notably, thrombospondin-2 (THBS2)). This process guides the mechanical remodelling of HSPCs via modulation of the actin cytoskeleton and the extracellular matrix, with THBS2 interacting with the integrin alpha v beta 3 and coordinating the nuclear translocation of YAP upon CRH/CRH-receptor-1 (CRH/CRHR1) signalling. Overall, the CRH/CRHR1/RhoA/YAP/THBS2/alpha v beta 3 axis has a central role in modulating HSPC behaviour via a mechanical feedback loop involving THBS2, alpha v beta 3, the actin cytoskeleton and YAP signalling. Our findings may suggest avenues for optimizing the transplantation of HSPCs.

Juvenile myelomonocytic leukemia (JMML), a clonal hematologic malignancy, originates from mutated hematopoietic stem cells (HSCs). The mechanism sustaining the persistence of mutant stem cells, leading to leukemia development, remains elusive. In this study, we conducted comprehensive examination of gene expression profiles, transcriptional factor regulons, and cell compositions/interactions throughout various stages of tumor cell development in Ptpn11 mutation-associated JMML. Our analyses revealed that leukemia-initiating Ptpn11 E76K/+ mutant stem cells exhibited de novo activation of the myeloid transcriptional program and aberrant developmental trajectories. These mutant stem cells displayed significantly elevated expression of innate immunity-associated anti-microbial peptides and pro-inflammatory proteins, particularly S100a9 and S100a8. Biological experiments confirmed that S100a9/S100a8 conferred a selective advantage to the leukemia-initiating cells through autocrine effects and facilitated immune evasion by recruiting and promoting immune suppressive myeloid-derived suppressor cells (MDSCs) in the microenvironment. Importantly, pharmacological inhibition of S100a9/S100a8 signaling effectively impeded leukemia development from Ptpn11 E76K/+ mutant stem cells. These findings collectively suggest that JMML tumor-initiating cells exploit evolutionarily conserved innate immune and inflammatory mechanisms to establish clonal dominance.

Mutations that decrease or increase the activity of the tyrosine phosphatase, SHP2 (encoded by PTPN11), promotes developmental disorders and several malignancies by varying phosphatase activity. We uncovered that SHP2 is a distinct class of an epigenetic enzyme; upon phosphorylation by the kinase ACK1/TNK2, pSHP2 was escorted by androgen receptor (AR) to chromatin, erasing hitherto unidentified pY54-H3 (phosphorylation of histones H3 at Tyr54) epigenetic marks to trigger a transcriptional program of AR. Noonan Syndrome with Multiple Lentigines (NSML) patients, SHP2 knock-in mice, and ACK1 knockout mice presented dramatic increase in pY54-H3, leading to loss of AR transcriptome. In contrast, prostate tumors with high pSHP2 and pACK1 activity exhibited progressive downregulation of pY54-H3 levels and higher AR expression that correlated with disease severity. Overall, pSHP2/pY54-H3 signaling acts as a sentinel of AR homeostasis, explaining not only growth retardation, genital abnormalities and infertility among NSML patients, but also significant AR upregulation in prostate cancer patients.

PDF file - 54K, Depletion of Shp2 results in lagging chromosomes during unperturbed (without nocodazole treatment) mitosis

PDF file - 39K, Shp2 depletion causes premature PLK1 and Cyclin B1 degradation

Background Cardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation. Methods hiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli. Results Treatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features—including enhanced Ca 2+ transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells. Conclusion AMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases.

PDF file - 27K, Shp2 depletion decreases taxol-induced mitotic arrest

While mitochondria in different tissues have distinct preferences for energy sources, they are flexible in utilizing competing substrates for metabolism according to physiological and nutritional circumstances. However, the regulatory mechanisms and significance of metabolic flexibility are not completely understood. Here we report that the deletion of PTPMT1, a mitochondria-based phosphatase, critically alters mitochondrial fuel selection – the utilization of pyruvate, a key mitochondrial substrate derived from glucose (the major simple carbohydrate), is inhibited, whereas the fatty acid utilization is enhanced. PTPMT1 knockout does not impact the development of the skeletal muscle or heart. However, the metabolic inflexibility ultimately leads to muscular atrophy, heart failure, and sudden death. Mechanistic analyses reveal that the prolonged substrate shift from carbohydrates to lipids causes oxidative stress and mitochondrial destruction, which in turn results in marked accumulation of lipids and profound damage in the knockout muscle cells and cardiomyocytes. Interestingly, PTPMT1 deletion from the liver or adipose tissue does not generate any local or systemic defects. These findings suggest that PTPMT1 plays an important role in maintaining mitochondrial flexibility and that their balanced utilization of carbohydrates and lipids is essential for both the skeletal muscle and the heart despite the two tissues having different preferred energy sources.

PDF file - 27K, Shp2-depleted cells fail to sustain nocodazole-induced mitotic arrest

PDF file - 80KB, Identification of active compounds that inhibited the phosphatase activity of SHP2 with various efficiencies.

PDF file - 111 KB, #220-324 inhibited SHP2-mediated cell signaling.

PDF file - 65 KB, Effect of Triton X-100 on the function of #220-324 in inhibiting SHP2 activity.