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Our data previously revealed that chemosurviving cancer cells translate specific genes. Here, we find that the m6A-RNA-methyltransferase, METTL3, increases transiently in chemotherapy-treated breast cancer and leukemic cells in vitro and in vivo. Consistently, m6A increases on RNA from chemo-treated cells, and is needed for chemosurvival. This is regulated by eIF2α phosphorylation and mTOR inhibition upon therapy treatment. METTL3 mRNA purification reveals that eIF3 promotes METTL3 translation that is reduced by mutating a 5′UTR m6A-motif or depleting METTL3. METTL3 increase is transient after therapy treatment, as metabolic enzymes that control methylation and thus m6A levels on METTL3 RNA, are altered over time after therapy. Increased METTL3 reduces proliferation and anti-viral immune response genes, and enhances invasion genes, which promote tumor survival. Consistently, overriding phospho-eIF2α prevents METTL3 elevation, and reduces chemosurvival and immune-cell migration. These data reveal that therapy-induced stress signals transiently upregulate METTL3 translation, to alter gene expression for tumor survival.One sentence summarym6A enzyme translation upon therapy stress, promotes tumor survival

Quiescent leukemic cells survive chemotherapy, with translation changes. Our data reveal that FXR1, a protein amplified in several aggressive cancers, is elevated in quiescent and chemo-treated leukemic cells and promotes chemosurvival. This suggests undiscovered roles for this RNA- and ribosome-associated protein in chemosurvival. We find that FXR1 depletion reduces translation, with altered rRNAs, snoRNAs, and ribosomal proteins (RPs). FXR1 regulates factors that promote transcription and processing of ribosomal genes and snoRNAs. Ribosome changes in FXR1-overexpressing cells, including RPLP0/uL10 levels, activate eIF2α kinases. Accordingly, phospho-eIF2α increases, enabling selective translation of survival and immune regulators in FXR1-overexpressing cells. Overriding these genes or phospho-eIF2α with inhibitors reduces chemosurvival. Thus, elevated FXR1 in quiescent or chemo-treated leukemic cells alters ribosomes that trigger stress signals to redirect translation for chemosurvival.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of unknown cause, characterized by infiltration and accumulation of activated immune cells in the synovial joints where cartilage and bone destructions occur. Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) was shown to be involved in the regulation of MDSC differentiation. The purpose of the present study was to investigate the effect of inhibition of SHIP1 on the expansion of MDSCs in RA using a collagen-induced inflammatory arthritis (CIA) mouse model. In DBA/1 mice, treatment with a small molecule-specific SHIP1 inhibitor 3α-aminocholestane (3AC) induced a marked expansion of MDSCs in vivo. Both pretreatment with 3AC of DBA/1 mice prior to CIA induction and intervention with 3AC during CIA progression significantly reduced disease incidence and severity. Adoptive transfer of MDSCs isolated from 3AC-treated mice, but not naïve MDSCs from normal mice, into CIA mice significantly reduced disease incidence and severity, indicating that the 3AC-induced MDSCs were the cellular mediators of the observed amelioration of the disease. In conclusion, inhibition of SHIP1 expands MDSCs in vivo and attenuates development of CIA in mice. Small molecule-specific inhibition of SHIP1 may therefore offer therapeutic benefit to patients with RA and other autoimmune diseases.

Adult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) ensuring homeostasis of blood production and immune response throughout life. Sex differences in immunocompetence and mortality are well-documented in humans. However, whether HSPCs behave dimorphically between sexes during aging remains unknown. Here, we show that a significant expansion of BM-derived HSPCs occurs in the middle age of female but in the old age of male mice. We then show that a decline of HSPCs in male mice, as indicated by the expression levels of select hematopoietic genes, occurs much earlier in the aging process than that in female mice. Sex-mismatched heterochronic BM transplantations indicate that the middle-aged female BM microenvironment plays a pivotal role in sustaining hematopoietic gene expression during aging. Furthermore, a higher concentration of the pituitary sex hormone follicle-stimulating hormone (FSH) in the serum and a concomitant higher expression of its receptor on HSPCs in the middle-aged and old female mice than age-matched male mice, suggests that FSH may contribute to the sexual dimorphism in aging hematopoiesis. Our study reveals that HSPCs in the BM niches are possibly regulated in a sex-specific manner and influenced differently by sex hormones during aging hematopoiesis.

Mesenchymal stem cell extracellular vesicles attenuate pulmonary hypertension, but their ability to reverse established disease in larger animal models and the duration and mechanism(s) of their effect are unknown. We sought to determine the efficacy and mechanism of mesenchymal stem cells’ extracellular vesicles in attenuating pulmonary hypertension in rats with Sugen/hypoxia-induced pulmonary hypertension. Male rats were treated with mesenchymal stem cell extracellular vesicles or an equal volume of saline vehicle by tail vein injection before or after subcutaneous injection of Sugen 5416 and exposure to 3 weeks of hypoxia. Pulmonary hypertension was assessed by right ventricular systolic pressure, right ventricular weight to left ventricle + septum weight, and muscularization of peripheral pulmonary vessels. Immunohistochemistry was used to measure macrophage activation state and recruitment to lung. Mesenchymal stem cell extracellular vesicles injected before or after induction of pulmonary hypertension normalized right ventricular pressure and reduced right ventricular hypertrophy and muscularization of peripheral pulmonary vessels. The effect was consistent over a range of doses and dosing intervals and was associated with lower numbers of lung macrophages, a higher ratio of alternatively to classically activated macrophages (M2/M1 = 2.00 ± 0.14 vs. 1.09 ± 0.11; P

Quiescent leukemic cells survive chemotherapy, with translation changes. Our data reveal that FXR1, a protein amplified in several aggressive cancers, increases in quiescent and chemo- treated leukemic cells, and promotes chemosurvival. This suggests undiscovered roles for this RNA- and ribosome-associated protein in chemosurvival. FXR1 depletion decreases translation and ribosome subunits, with altered rRNAs, snoRNAs, and ribosomal proteins (RPs). We find that FXR1 binds factors that promote ribosome gene transcription and bind snoRNAs. Ribosome changes increased in FXR1-overexpressing cells, including increased snoRNAs and RPLP0/uL10, activate eIF2α kinases. Accordingly, phospho-eIF2α increases, enabling non- canonical translation of survival and immune regulators in FXR1-overexpressing cells. Overriding these with inhibitors reduces chemosurvival. Thus, increased FXR1 in quiescent or chemo-treated leukemic cells, alters ribosomes that trigger stress signals to re-direct translation for chemosurvival.One Sentence SummaryFXR1 alters ribosomes in G0, which induce stress signals to elicit noncanonical translation for AML drug and immune survival.

SH2‐containing inositol‐5′‐phosphatase‐1 (SHIP‐1) controls the phosphatidylinositol‐3′‐kinase (PI3K) initiated signaling pathway by limiting cell membrane recruitment and activation of Akt. Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the PI3K signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis. Here, we report that the lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development through its expression in osteochondroprogenitor cells. Global SHIP‐1 knockout led to accelerated chondrocyte hypertrophy and premature formation of the secondary ossification center in the bones of postnatal mice. Drastically higher vascularization and greater number of c‐kit + progenitors associated with sinusoids in the bone marrow also indicated more advanced chondrocyte hypertrophic differentiation in SHIP‐1 knockout mice than in wild‐type mice. In corroboration with the in vivo phenotype, SHIP‐1 deficient PDGFRα + Sca‐1 + osteochondroprogenitor cells exhibited rapid differentiation into hypertrophic chondrocytes under chondrogenic culture conditions in vitro. Furthermore, SHIP‐1 deficiency inhibited hypoxia‐induced cellular activation of Akt and extracellular‐signal‐regulated kinase (Erk) and suppressed hypoxia‐induced cell proliferation. These results suggest that SHIP‐1 is required for hypoxia‐induced growth signaling under physiological hypoxia in the bone marrow. In conclusion, the lipid phosphatase SHIP‐1 regulates skeletal development by modulating chondrogenesis and the hypoxia response of the osteochondroprogenitors during endochondral bone formation., In this study, we found that lipid phosphatase SH2‐containing inositol‐5′‐phosphatase‐1 (SHIP‐1) negatively regulates the hypertrophic process of growth plate chondrocytes. SHIP‐1 deficiency accelerates the formation of the secondary ossification center, and SHIP‐1 KO mice have elevated vascularization and more c‐kit + progenitor cells in the bone marrow compartment. SHIP‐1 is also required for hypoxia‐induced Akt/Erk activation and cell proliferation.

Aims Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. Previously, we found that transcription factor RUNX1-dependent haematopoietic transformation of endothelial progenitor cells may contribute to the pathogenesis of PAH. However, the therapeutic potential of RUNX1 inhibition to reverse established PAH remains unknown. In the current study, we aimed to determine whether RUNX1 inhibition was sufficient to reverse Sugen/hypoxia (SuHx)-induced pulmonary hypertension (PH) in rats. We also aimed to demonstrate possible mechanisms involved. Methods and results We administered a small molecule specific RUNX1 inhibitor Ro5-3335 before, during, and after the development of SuHx-PH in rats to investigate its therapeutic potential. We quantified lung macrophage recruitment and activation in vivo and in vitro in the presence or absence of the RUNX1 inhibitor. We generated conditional VE-cadherin-CreERT2; ZsGreen mice for labelling adult endothelium and lineage tracing in the SuHx-PH model. We also generated conditional Cdh5-CreERT2; Runx1(flox/flox) mice to delete Runx1 gene in adult endothelium and LysM-Cre; Runx1(flox/flox) mice to delete Runx1 gene in cells of myeloid lineage, and then subjected these mice to SuHx-PH induction. RUNX1 inhibition in vivo effectively prevented the development, blocked the progression, and reversed established SuHx-induced PH in rats. RUNX1 inhibition significantly dampened lung macrophage recruitment and activation. Furthermore, lineage tracing with the inducible VE-cadherin-CreERT2; ZsGreen mice demonstrated that a RUNX1-dependent endothelial to haematopoietic transformation occurred during the development of SuHx-PH. Finally, tissue-specific deletion of Runx1 gene either in adult endothelium or in cells of myeloid lineage prevented the mice from developing SuHx-PH, suggesting that RUNX1 is required for the development of PH. Conclusion By blocking RUNX1-dependent endothelial to haematopoietic transformation and pulmonary macrophage recruitment and activation, targeting RUNX1 may be as a novel treatment modality for pulmonary arterial hypertension.

Infantile hemangiomas are benign tumors of vascular endothelial cells (ECs), characterized by three distinct stages: proliferating phase, involuting phase, and involuted phase. The mechanisms that trigger involution of hemangioma into fibro-fatty tissue remain unknown. We report a novel mechanism by which M1-polarized macrophages induce endothelial-to-mesenchymal transition (EndMT) and promote hemangioma regression. M1- but not M2-polarized macrophages induced EndMT in ECs. Tumor necrosis factor-α and, to a lesser extent, IL-1β and interferon-γ were the most potent cytokines produced by the M1 macrophages that induce in vitro EndMT. Western blot analysis and gene expression profiling showed that ECs treated with M1 macrophages, tumor necrosis factor-α, or IL-1β decreased the expression of endothelial markers, whereas mesenchymal markers increased concomitantly. Immunohistochemical staining of patient samples revealed that a significant perivascular infiltration of M1, but not M2, macrophages coincides with endothelial expression of the critical EndMT transcription factors Snail/Slug in involuting hemangiomas. Most strikingly, M1 macrophage–treated ECs isolated from patient hemangiomas (HemECs) but not untreated HemECs readily differentiated into adipocytes on adipogenic induction. Thus, in vitro EndMT and adipogenesis of HemECs have, in part, recapitulated the natural history of hemangioma regression. In conclusion, our findings indicate that EndMT induced by M1 macrophages promotes infantile hemangioma regression and may lead to novel therapeutic treatments for this vascular tumor.

Aims The pathogenic mechanisms of pulmonary arterial hypertension (PAH) remain unclear, but involve dysfunctional endothelial cells (ECs), dysregulated immunity and inflammation in the lung. We hypothesize that a developmental process called endothelial to haematopoietic transition (EHT) contributes to the pathogenesis of pulmonary hypertension (PH). We sought to determine the role of EHT in mouse models of PH, to characterize specific cell types involved in this process, and to identify potential therapeutic targets to prevent disease progression. Methods and results When transgenic mice with fluorescence protein ZsGreen-labelled ECs were treated with Sugen/hypoxia (Su/Hx) combination to induce PH, the percentage of ZsGreen+ haematopoietic cells in the peripheral blood, primarily of myeloid lineage, significantly increased. This occurrence coincided with the depletion of bone marrow (BM) ZsGreen+ c-kit+ CD45- endothelial progenitor cells (EPCs), which could be detected accumulating in the lung upon PH-induction. Quantitative RT-PCR based gene array analysis showed that key transcription factors driving haematopoiesis were expressed in these EPCs. When transplanted into lethally irradiated recipient mice, the BM-derived EPCs exhibited long-term engraftment and haematopoietic differentiation capability, indicating these EPCs are haemogenic in nature. Specific inhibition of the critical haematopoietic transcription factor Runx1 blocked the EHT process in vivo, prevented egress of the BM EPCs and ultimately attenuated PH progression in Su/Hx- as well as in monocrotaline-induced PH in mice. Thus, myeloid-skewed EHT promotes the development of PH and inhibition of this process prevents disease progression in mouse models of PH. Furthermore, high levels of Runx1 expression were found in circulating CD34+ CD133+ EPCs isolated from peripheral blood of patients with PH, supporting the clinical relevance of our proposed mechanism of EHT. Conclusion EHT contributes to the pathogenesis of PAH. The transcription factor Runx1 may be a novel therapeutic target for the treatment of PAH.