SHP-1 inhibition targets leukaemia stem cells to restore immunosurveillance and enhance chemosensitivity by metabolic reprogramming.

Leukaemia stem cells (LSCs) in acute myeloid leukaemia present a considerable treatment challenge due to their resistance to chemotherapy and immunosurveillance. The connection between these properties in LSCs remains poorly understood. Here we demonstrate that inhibition of tyrosine phosphatase SHP-1 in LSCs increases their glycolysis and oxidative phosphorylation, enhancing their sensitivity to chemotherapy and vulnerability to immunosurveillance. Mechanistically, SHP-1 inhibition leads to the upregulation of phosphofructokinase platelet (PFKP) through the AKT-ß-catenin pathway. The increase in PFKP elevates energy metabolic activities and, as a consequence, enhances the sensitivity of LSCs to chemotherapeutic agents. Moreover, the upregulation of PFKP promotes MYC degradation and, consequently, reduces the immune evasion abilities of LSCs. Overall, our study demonstrates that targeting SHP-1 disrupts the metabolic balance in LSCs, thereby increasing their vulnerability to chemotherapy and immunosurveillance. This approach offers a promising strategy to overcome LSC resistance in acute myeloid leukaemia.

A Ferroptosis-Inducing and Leukemic Cell-Targeting Drug Nanocarrier Formed by Redox-Responsive Cysteine Polymer for Acute Myeloid Leukemia Therapy.

Ferroptosis is an alternative strategy to overcome chemoresistance, but effective therapeutic approaches to induce ferroptosis for acute myeloid leukemia (AML) treatment are limited. Here, we developed glutathione (GSH)-responsive cysteine polymer-based ferroptosis-inducing nanomedicine (GCFN) as an efficient ferroptosis inducer and chemotherapeutic drug nanocarrier for AML treatment. GCFN depleted intracellular GSH and inhibited glutathione peroxidase 4, a GSH-dependent hydroperoxidase, to cause lipid peroxidation and ferroptosis in AML cells. Furthermore, GCFN-loaded paclitaxel (PTX@GCFN) targeted AML cells and spared normal hematopoietic cells to limit the myeloablation side effects caused by paclitaxel. PTX@GCFN treatment extended the survival of AML mice by specifically releasing paclitaxel and simultaneously inducing ferroptosis in AML cells with restricted myeloablation and tissue damage side effects. Overall, the dual-functional GCFN acts as an effective ferroptosis inducer and a chemotherapeutic drug carrier for AML treatment.

PD-1 signalling defines and protects leukaemic stem cells from T cell receptor-induced cell death in T cell acute lymphoblastic leukaemia.

T cell acute lymphoblastic leukaemia (T-ALL) is an aggressive malignancy with poor prognosis, but a decisive marker and effective treatment for leukaemia stem cells (LSCs) remain unclear. Here, using lineage tracing, limiting dilution assays and in vivo live imaging approaches, we identify rare inhibitory receptor programmed cell death 1 (PD-1)-expressing cells that reside at the apex of leukaemia hierarchy for initiation and relapse in T-ALL. Ablation of PD-1-expressing cells, deletion of PD-1 in T-ALL cells or blockade of PD-1 or PD-1 ligand 1 significantly eradicated LSCs and suppressed disease progression. Combination therapy using PD-1 blockade and chemotherapy substantially extended the survival of mice engrafted with mouse or human T-ALL cells. Mechanistically, PD-1+ LSCs had high NOTCH1-MYC activity for disease initiation. Furthermore, PD-1 signalling maintained quiescence and protected LSCs against T cell receptor-signal-induced apoptosis. Overall, our data highlight the hierarchy of leukaemia by identifying PD-1+ LSCs and provide a therapeutic approach for the elimination of LSCs through PD-1 blockade in T-ALL.

A small secreted protein, RsMf8HN, in Rhizoctonia solani triggers plant immune response, which interacts with rice OsHIPP28.

The necrotrophic phytopathogen Rhizoctonia solani (R. solani) causes disease in many plant species. This fungal genome encodes abundant small cysteine-rich (SCR)-secreted proteins in R. solani that may induce pathogenesis. To test their molecular functions, we introduced 10 SCR-secreted protein genes from R. solani into tobacco leaves via agroinfiltration. Consequently, we identified RsMf8HN, a novel SCR protein that triggers cell death and an oxidative burst in tobacco. RsMf8HN comprises 182 amino acids (aa), including a signal peptide (SP) of 17aa, and the protein has unique features: it is orthologous to an allergen protein Mal f 8 occurring in Malassezia species, and possesses a high glycine and serine content. RsMf8HN is coded in a genomic location along with its paralogues and a few other effector candidates. The elicitation of plant immunity by RsMf8HN was dependent on HSP90 and SGT1. RsMf8HN was translocated to multiple locations within the host cells: i.e., nuclei, chloroplasts, and plasma membranes. We confirmed the occurrence of in vivo cross-interactions of RsMf8HN with a rice molecule, the heavy metal-associated isoprenylated plant protein OsHIPP28, which is a protein related to the disease susceptibility factor Pi21. In summary, our results suggest that RsMf8HN is a potential effector that enables necrotrophic phytopathogens to interfere with host plant immunity.

Amino acid catabolism regulates hematopoietic stem cell proteostasis via a GCN2-eIF2α axis.

Hematopoietic stem cells (HSCs) adapt their metabolism to maintenance and proliferation; however, the mechanism remains incompletely understood. Here, we demonstrated that homeostatic HSCs exhibited high amino acid (AA) catabolism to reduce cellular AA levels, which activated the GCN2-eIF2α axis, a protein synthesis inhibitory checkpoint to restrain protein synthesis for maintenance. Furthermore, upon proliferation conditions, HSCs enhanced mitochondrial oxidative phosphorylation (OXPHOS) for higher energy production but decreased AA catabolism to accumulate cellular AAs, which inactivated the GCN2-eIF2α axis to increase protein synthesis and coupled with proteotoxic stress. Importantly, GCN2 deletion impaired HSC function in repopulation and regeneration. Mechanistically, GCN2 maintained proteostasis and inhibited Src-mediated AKT activation to repress mitochondrial OXPHOS in HSCs. Moreover, the glycolytic metabolite, NAD+ precursor nicotinamide riboside (NR), accelerated AA catabolism to activate GCN2 and sustain the long-term function of HSCs. Overall, our study uncovered direct links between metabolic alterations and translation control in HSCs during homeostasis and proliferation.

Loss of sphingosine kinase 2 promotes the expansion of hematopoietic stem cells by improving their metabolic fitness.

Hematopoietic stem cells (HSCs) have reduced capacities to properly maintain and replenish the hematopoietic system during myelosuppressive injury or aging. Expanding and rejuvenating HSCs for therapeutic purposes has been a long-sought goal with limited progress. Here, we show that the enzyme Sphk2 (sphingosine kinase 2), which generates the lipid metabolite sphingosine-1-phosphate, is highly expressed in HSCs. The deletion of Sphk2 markedly promotes self-renewal and increases the regenerative potential of HSCs. More importantly, Sphk2 deletion globally preserves the young HSC gene expression pattern, improves the function, and sustains the multilineage potential of HSCs during aging. Mechanistically, Sphk2 interacts with prolyl hydroxylase 2 and the Von Hippel-Lindau protein to facilitate HIF1α ubiquitination in the nucleus independent of the Sphk2 catalytic activity. Deletion of Sphk2 increases hypoxic responses by stabilizing the HIF1α protein to upregulate PDK3, a glycolysis checkpoint protein for HSC quiescence, which subsequently enhances the function of HSCs by improving their metabolic fitness; specifically, it enhances anaerobic glycolysis but suppresses mitochondrial oxidative phosphorylation and generation of reactive oxygen species. Overall, targeting Sphk2 to enhance the metabolic fitness of HSCs is a promising strategy to expand and rejuvenate functional HSCs.

CXCR4high megakaryocytes regulate host-defense immunity against bacterial pathogens.

Megakaryocytes (MKs) continuously produce platelets to support hemostasis and form a niche for hematopoietic stem cell maintenance in the bone marrow. MKs are also involved in inflammatory responses; however, the mechanism remains poorly understood. Using single-cell sequencing, we identified a CXCR4 highly expressed MK subpopulation, which exhibited both MK-specific and immune characteristics. CXCR4high MKs interacted with myeloid cells to promote their migration and stimulate the bacterial phagocytosis of macrophages and neutrophils by producing TNFα and IL-6. CXCR4high MKs were also capable of phagocytosis, processing, and presenting antigens to activate T cells. Furthermore, CXCR4high MKs also egressed circulation and infiltrated into the spleen, liver, and lung upon bacterial infection. Ablation of MKs suppressed the innate immune response and T cell activation to impair the anti-bacterial effects in mice under the Listeria monocytogenes challenge. Using hematopoietic stem/progenitor cell lineage-tracing mouse lines, we show that CXCR4high MKs were generated from infection-induced emergency megakaryopoiesis in response to bacterial infection. Overall, we identify the CXCR4high MKs, which regulate host-defense immune response against bacterial infection.

A self-assembled leucine polymer sensitizes leukemic stem cells to chemotherapy by inhibiting autophagy in acute myeloid leukemia.

Chemotherapy is the primary treatment option for acute myeloid leukemia (AML), but leukemic stem cells (LSC) can survive chemotherapy for disease recurrence and refractory. Here, we found that AML cells obtained from relapsed patients had increased autophagy levels than de novo AML cells. Furthermore, doxorubicin (DOX) treatment stimulated autophagy in LSC by repressing the mTOR pathway, and pharmaceutical inhibition of autophagy rendered chemoresistant LSC sensitive to DOX treatment in MLL-AF9 induced murine AML. Moreover, we developed a self-assembled leucine polymer, which activated mTOR to inhibit autophagy in AML cells by releasing leucine. The leucine polymer loaded DOX (Leu-DOX) induced much less autophagy but more robust apoptosis in AML cells than the DOX treatment. Notably, the leucine polymer and Leu-DOX were specifically taken up by AML cells and LSC but not by normal hematopoietic cells and hematopoietic stem/progenitor cells in the bone marrow. Consequently, Leu-DOX efficiently reduced LSC and prolonged the survival of AML mice, with more limited myeloablation and tissue damage side effects than DOX treatment. Overall, we proposed that the newly developed Leu-DOX is an effective autophagy inhibitor and an ideal drug to efficiently eliminate LSC, thus serving as a revolutionary strategy to enhance the chemotherapy efficacy in AML.

The RIG-I-NRF2 axis regulates the mesenchymal stromal niche for bone marrow transplantation.

Bone marrow-derived mesenchymal stem cells (BMSCs) support bone formation and constitute the stromal niche in regulating hematopoietic stem cells (HSCs). Stromal niche dysfunction affects HSC engraftment during transplantation; however, the underlying mechanisms remain elusive. In the present study, we found that all-trans retinoic acid (ATRA) and inflammation stress upregulated retinoic acid-inducible gene I (RIG-I) in BMSCs. Excess RIG-I expression damaged the clonogenicity, bone-forming ability of BMSCs and particularly their stromal niche function that supports HSC expansion in vitro and engraftment in vivo. Mechanistically, RIG-I elevation promoted the degradation of NRF2, a checkpoint for antioxidant cellular response, by altering the RIG-I-Trim25-Keap1-NRF2 complex, leading to reactive oxygen species (ROS) accumulation and BMSC damage. Genetic inhibition of RIG-I sustained NRF2 protein levels and reduced ROS levels in ATRA-treated BMSCs, thus preserving their clonogenicity, bone-forming ability, and stromal niche function in supporting HSC engraftment in mice. More importantly, RIG-I inhibition recovered the ATRA-treated stromal niche function to enhance HSC engraftment and emergency myelopoiesis for innate immunity against the bacterium Listeria monocytogenes during transplantation. Overall, we identified a noncanonical role of RIG-I in the regulation of the stromal niche for HSC transplantation.

A Metabolic Reprogramming Amino Acid Polymer as an Immunosurveillance Activator and Leukemia Targeting Drug Carrier for T-Cell Acute Lymphoblastic Leukemia.

Compromised immunosurveillance leads to chemotherapy resistance and disease relapse of hematological malignancies. Amino acid metabolism regulates immune responses and cancer; however, a druggable amino acid metabolite to enhance antitumor immunosurveillance and improve leukemia targeting-therapy efficacy remains unexplored. Here, an L-phenylalanine polymer, Metabolic Reprogramming Immunosurveillance Activation Nanomedicine (MRIAN), is invented to effectively target bone marrow (BM) and activate the immune surveillance in T-cell acute lymphoblastic leukemia (T-ALL) by inhibiting myeloid-derived suppressor cells (MDSCs) in T-ALL murine model. Stable-isotope tracer and in vivo drug distribution experiments show that T-ALL cells and MDSCs have enhanced cellular uptake of L-phenylalanine and MRIANs than normal hematopoietic cells and progenitors. Therefore, MRIAN assembled Doxorubicin (MRIAN-Dox) specifically targets T-ALL cells and MDSCs but spare normal hematopoietic cells and hematopoietic stem and progenitor cells with enhanced leukemic elimination efficiency. Consequently, MRIAN-Dox has reduced cardiotoxicity and myeloablation side effects in treating T-ALL mice. Mechanistically, MRIAN degrades into L-phenylalanine, which inhibits PKM2 activity and reduces ROS levels in MDSCs to disturb their immunosuppressive function and increase their differentiation toward normal myeloid cells. Overall, a novel amino acid metabolite nanomedicine is invented to treat T-ALL through the combination of leukemic cell targeting and immunosurveillance stimulation.

A lysosome-targeted dextran-doxorubicin nanodrug overcomes doxorubicin-induced chemoresistance of myeloid leukemia.

The hypoxic microenvironment is presumed to be a sanctuary for myeloid leukemia cells that causes relapse following chemotherapy, but the underlying mechanism remains elusive. Using a zebrafish xenograft model, we observed that the hypoxic hematopoietic tissue preserved most of the chemoresistant leukemic cells following the doxorubicin (Dox) treatment. And hypoxia upregulated TFEB, a master regulator of lysosomal biogenesis, and increased lysosomes in leukemic cells. Specimens from relapsed myeloid leukemia patients also harbored excessive lysosomes, which trapped Dox and prevented drug nuclear influx leading to leukemia chemoresistance. Pharmaceutical inhibition of lysosomes enhanced Dox-induced cytotoxicity against leukemic cells under hypoxia circumstance. To overcome lysosome associated chemoresistance, we developed a pH-sensitive dextran-doxorubicin nanomedicine (Dex-Dox) that efficiently released Dox from lysosomes and increased drug nuclear influx. More importantly, Dex-Dox treatment significantly improved the chemotherapy outcome in the zebrafish xenografts transplanted with cultured leukemic cells or relapsed patient specimens. Overall, we developed a novel lysosome targeting nanomedicine that is promising to overcome the myeloid leukemia chemoresistance.

Retinoic Acid Inhibits Tumor-Associated Mesenchymal Stromal Cell Transformation in Melanoma.

Bone marrow mesenchymal stem/stromal cells (BMSCs) can be transformed into tumor-associated MSCs (TA-MSCs) within the tumor microenvironment to facilitate tumor progression. However, the underline mechanism and potential therapeutic strategy remain unclear. Here, we explored that interleukin 17 (IL-17) cooperating with IFNγ transforms BMSCs into TA-MSCs, which promotes tumor progression by recruiting macrophages/monocytes and myeloid-derived suppressor cells (MDSCs) in murine melanoma. IL-17 and IFNγ transformed TA-MSCs have high expression levels of myelocyte-recruiting chemokines (CCL2, CCL5, CCL7, and CCL20) mediated by activated NF-κB signaling pathway. Furthermore, retinoic acid inhibits NF-κB signaling, decreases chemokine expression, and suppresses the tumor-promoting function of transformed TA-MSCs by prohibiting the recruitment of macrophages/monocytes and MDSCs in the tumor microenvironment. Overall, our findings demonstrate that IL-17 collaborating with IFNγ to induce TA-MSC transformation, which can be targeted by RA for melanoma treatment.

Single-Cell Atlas Reveals Fatty Acid Metabolites Regulate the Functional Heterogeneity of Mesenchymal Stem Cells.

Bone marrow mesenchymal stem cells (MSCs) are widely used clinically due to their versatile roles in multipotency, immunomodulation, and hematopoietic stem cell (HSC) niche function. However, cellular heterogeneity limits MSCs in the consistency and efficacy of their clinical applications. Metabolism regulates stem cell function and fate decision; however, how metabolites regulate the functional heterogeneity of MSCs remains elusive. Here, using single-cell RNA sequencing, we discovered that fatty acid pathways are involved in the regulation of lineage commitment and functional heterogeneity of MSCs. Functional assays showed that a fatty acid metabolite, butyrate, suppressed the self-renewal, adipogenesis, and osteogenesis differentiation potential of MSCs with increased apoptosis. Conversely, butyrate supplement significantly promoted HSC niche factor expression in MSCs, which suggests that butyrate supplement may provide a therapeutic approach to enhance their HSC niche function. Overall, our work demonstrates that metabolites are essential to regulate the functional heterogeneity of MSCs.

Multifunctional sharp pH-responsive nanoparticles for targeted drug delivery and effective breast cancer therapy.

The intrinsic limits of conventional cancer therapies prompt the development of a new technology for a more effective and safer cancer treatment. The bioresponsive delivery technique has recently emerged as an innovative strategy to overcome multiple barriers in the systemic delivery of nanoparticle (NP)-based therapeutics. However, some issues especially the tumor penetration-retention balance have not been completely solved, which may induce the suboptimal therapeutic effect. Herein, we developed a new multifunctional sharp pH-responsive NP platform for targeted drug delivery and effective cancer therapy. This NP platform is made of the sharp pH-responsive poly(2-(diisopropylamino)ethylmethacrylate) (PDPA) polymer as the inner core, amphiphilic lipid-poly(ethylene glycol) (lipid-PEG) as the outer shell, and the internalizing RGD (iRGD) peptide encoded on the surface. After anticancer drug loading and then systemic administration, the resulting NP platform shows the following features in one nanostructure: (i) the PEG shell to prolong blood circulation; (ii) the iRGD peptide to enhance tumor targeting and penetration; (iii) a larger particle size (∼80 nm) than that of free drug to ensure long tumor retention; (iv) the sharp endosomal pH response of the PDPA polymer to induce fast intracellular drug release and thus efficient inhibition of tumor growth. Together with facile polymer synthesis and robust NP formulation to enable easy scale-up, the multifunctional NP platform reported herein shows great potential as a new generation nanomedicine for effective cancer treatment.

SHP-1 regulates hematopoietic stem cell quiescence by coordinating TGF-ß signaling.

Cell cycle quiescence is critical for hematopoietic stem cell (HSC) maintenance. TGF-ß signaling in bone marrow niche has been identified in regulating HSC quiescence; however, the intrinsic regulatory mechanisms remain unclear. This study reports that Shp-1 knockout HSCs have attenuated quiescence and impaired long-term self-renewal. SHP-1-activated HSCs are surrounded by megakaryocytes, which regulate HSC quiescence by producing TGF-ß1. Mechanistically, SHP-1 interacts with the immunoreceptor tyrosine-based inhibition motif on TGF-ß receptor 1 and is critical for TGF-ß signaling activation in HSCs. Functionally, Shp-1 knockout HSCs do not respond to TGF-ß-enforced HSC quiescence regulation, both in vitro and in vivo. Therefore, we identify TGF-ß-SHP-1 as a novel intrinsic regulatory mechanism for HSC quiescence maintenance.

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line.

Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/ß-catenin signaling following hair cell death. We propose that Wnt/ß-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals.

RIG-I modulates Src-mediated AKT activation to restrain leukemic stemness.

Retinoic acid (RA)-inducible gene I (RIG-I) is highly upregulated and functionally implicated in the RA-induced maturation of acute myeloid leukemia (AML) blasts. However, the underlying mechanism and the biological relevance of RIG-I expression to the maintenance of leukemogenic potential are poorly understood. Here, we show that RIG-I, without priming by foreign RNA, inhibits the Src-facilitated activation of AKT-mTOR in AML cells. Moreover, in a group of primary human AML blasts, RIG-I reduction renders the Src family kinases hyperactive in promoting AKT activation. Mechanistically, a PxxP motif in RIG-I, upon the N-terminal CARDs' association with the Src SH1 domain, competes with the AKT PxxP motif for recognizing the Src SH3 domain. In accordance, mutating PxxP motif prevents Rig-I from inhibiting AKT activation, cytokine-stimulated myeloid progenitor proliferation, and in vivo repopulating capacity of leukemia cells. Collectively, our data suggest an antileukemia activity of RIG-I via competitively inhibiting Src/AKT association.

RA-inducible gene-I induction augments STAT1 activation to inhibit leukemia cell proliferation.

RA-inducible gene I (RIG-I/DDX58) has been shown to activate IFN-ß promoter stimulator 1 (IPS-1) on recognizing cytoplasmic viral RNAs. It is unclear how RIG-I functions within the IFN and/or RA signaling process in acute myeloid leukemia (AML) cells, however, where obvious RIG-I induction is observed. Here, we show that the RIG-I induction functionally contributes to IFN-α plus RA-triggered growth inhibition of AML cells. Interestingly, although RIG-I induction itself is under the regulation of STAT1, a major IFN intracellular signal mediator, under circumstances in which it does not stimulate IPS-1, it conversely augments STAT1 activation to induce IFN-stimulatory gene expression and inhibit leukemia cell proliferation. Thus, our results unveil a previously undescribed RIG-I activity in regulating the cellular proliferation of leukemia cells via STAT1, which is independent of its classic role of sensing viral invasion to trigger type I IFN transcription.

RIG-I plays a critical role in negatively regulating granulocytic proliferation.

RIG-I has been implicated in innate immunity by sensing intracellular viral RNAs and inducing type I IFN production. However, we have found a significant RIG-I induction in a biological setting without active viral infection-namely, during RA-induced terminal granulocytic differentiation of acute myeloid leukemias. Here, we present evidence that a significant Rig-I induction also occurs during normal myelopoiesis and that the disruption of the Rig-I gene in mice leads to the development of a progressive myeloproliferative disorder. The initiation of progressive myeloproliferative disorder is mainly due to an intrinsic defect of Rig-I(-/-) myeloid cells, which are characterized by a reduced expression of IFN consensus sequence binding protein, a major regulator of myeloid differentiation. Thus, our study reveals a critical regulatory role of Rig-I in modulating the generation and differentiation of granulocytes.