MFSD7C protects hemolysis-induced lung impairments by inhibiting ferroptosis.

Hemolysis drives susceptibility to lung injury and predicts poor outcomes in diseases, such as malaria and sickle cell disease (SCD). However, the underlying pathological mechanism remains elusive. Here, we report that major facilitator superfamily domain containing 7 C (MFSD7C) protects the lung from hemolytic-induced damage by preventing ferroptosis. Mechanistically, MFSD7C deficiency in HuLEC-5A cells leads to mitochondrial dysfunction, lipid remodeling and dysregulation of ACSL4 and GPX4, thereby enhancing lipid peroxidation and promoting ferroptosis. Furthermore, systemic administration of MFSD7C mRNA-loaded nanoparticles effectively prevents lung injury in hemolytic mice, such as HbSS-Townes mice and PHZ-challenged 7 C-/- mice. These findings present the detailed link between hemolytic complications and ferroptosis, providing potential therapeutic targets for patients with hemolytic disorders.

Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation.

Here we explored the role of interleukin-1ß (IL-1ß) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1ß monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1ß/IL-1rn levels under steady-state, and that loss of repression of IL-1ß signaling may underlie pre-leukemic lesion and AML progression.

Role of ASXL1 in hematopoiesis and myeloid diseases.

Next-generation sequencing technology, including whole-exome or whole-genome sequencing and target gene sequencing, has allowed the molecular characterization of somatic mutation spectrums in hematologic diseases. Mutations in Additional sex combs-like 1 (ASXL1), a chromatin regulator, are identified in clonal hematopoiesis of indeterminate potential (CHIP), indicating ASXL1 mutations as early events in leukemogenesis. Not surprisingly, they occur at high frequency in myeloid malignancies and are associated with poor prognosis. Therefore, understanding how mutant ASXL1 drives clonal expansion and leukemogenesis will serve as the basis for the future development of preventative and/or therapeutic strategies for myeloid diseases with ASXL1 mutations. Here, we discuss the biology of ASXL1 and its role in controlling normal and malignant hematopoiesis. In addition, we review the clinical relevance of ASXL1 mutations in CHIP and myeloid diseases.

Ponatinib sensitizes myeloma cells to MEK inhibition in the high-risk VQ model.

Multiple myeloma (MM) is a malignant plasma cell cancer. Mutations in RAS pathway genes are prevalent in advanced and proteasome inhibitor (PI) refractory MM. As such, we recently developed a VQ MM mouse model recapitulating human advanced/high-risk MM. Using VQ MM cell lines we conducted a repurposing screen of 147 FDA-approved anti-cancer drugs with or without trametinib (Tra), a MEK inhibitor. Consistent with its high-risk molecular feature, VQ MM displayed reduced responses to PIs and de novo resistance to the BCL2 inhibitor, venetoclax. Ponatinib (Pon) is the only tyrosine kinase inhibitor that showed moderate MM killing activity as a single agent and strong synergism with Tra in vitro. Combined Tra and Pon treatment significantly prolonged the survival of VQ MM mice regardless of treatment schemes. However, this survival benefit was moderate compared to that of Tra alone. Further testing of Tra and Pon on cytotoxic CD8+ T cells showed that Pon, but not Tra, blocked T cell function in vitro, suggesting that the negative impact of Pon on T cells may partially counteract its MM-killing synergism with Tra in vivo. Our study provides strong rational to comprehensively evaluate agents on both MM cells and anti-MM immune cells during therapy development.

Inhaled mRNA Nanoformulation with Biogenic Ribosomal Protein Reverses Established Pulmonary Fibrosis in a Bleomycin-Induced Murine Model.

Idiopathic pulmonary fibrosis (IPF), a lethal respiratory disease with few treatment options, occurs due to repetitive microinjuries to alveolar epithelial cells (AECs) and progresses with an overwhelming deposition of extracellular matrix (ECM), ultimately resulting in fibrotic scars and destroyed the alveolar architecture. Here, an inhaled ribosomal protein-based mRNA nanoformulation is reported for clearing the intrapulmonary ECM and re-epithelializing the disrupted alveolar epithelium, thereby reversing established fibrotic foci in IPF. The nanoformulation is sequentially assembled by a ribosomal protein-condensed mRNA core, a bifunctional peptide-modified corona and keratinocyte growth factor (KGF) with a PEGylated shielding shell. When inhaled via a nebulizer, the nanoformulations carried by microdrops are deposited in the alveoli, and penetrate into fibrotic foci, where the outer KGFs are detached after matrix metalloproteinase 2 (MMP2) triggering. The RGD motif-grafted cores then expose and specifically target the integrin-elevated cells for the intracellular delivery of mRNA. Notably, repeated inhalation of the nanoformulations accelerates the clearance of locoregional collagen by boosting the intralesional expression of MMP13 and alveolar re-epithelialization mediated by KGFs, which synergistically ameliorates the lung function of a bleomycin-induced murine model. Therefore, this work provides an alternative mRNA-inhalation delivery strategy, which shows great potential for the treatment of IPF.

Asxl1 loss cooperates with oncogenic Nras in mice to reprogram the immune microenvironment and drive leukemic transformation.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

Gata2 +9.5 enhancer regulates adult hematopoietic stem cell self-renewal and T-cell development.

Mammalian GATA2 gene encodes a dual zinc finger transcription factor, which is essential for hematopoietic stem cell (HSC) generation in the aorta, gonad, mesonephros (AGM) region, HSC self-renewal, and specification of progenitor cell fates. Previously, we demonstrated that Gata2 expression in AGM is controlled by its intronic +9.5 enhancer. Gata2 +9.5 deficiency removes the E-box motif and the GATA site and depletes fetal liver HSCs. However, whether this enhancer has an essential role in regulating adult hematopoiesis has not been established. Here, we evaluate Gata2 +9.5 enhancer function in adult hematopoiesis. +9.5+/- bone marrow cells displayed reduced T cell reconstitution in a competitive transplant assay. Donor-derived analysis demonstrated a previously unrecognized function of the +9.5 enhancer in T cell development at the lymphoid-primed multipotent progenitor stage. Moreover, +9.5+/- adult HSCs displayed increased apoptosis and reduced long-term self-renewal capability in comparison with wild-type (WT) HSCs. These phenotypes were more moderate than those of Gata2+/- HSCs. Consistent with the phenotypic characterization, Gata2 expression in +9.5+/- LSKs was moderately higher than that in Gata2+/- LSKs, but lower than that in WT LSKs. Our data suggest that +9.5 deficiency compromises, without completely abrogating, Gata2 expression in adult HSCs.

RAS mutations drive proliferative chronic myelomonocytic leukemia via a KMT2A-PLK1 axis.

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

Embryonic Expression of NrasG 12 D Leads to Embryonic Lethality and Cardiac Defects.

Ras proteins control a complex intracellular signaling network. Gain-of-function mutations in RAS genes lead to RASopathy disorders in humans, including Noonan syndrome (NS). NS is the second most common syndromic cause of congenital heart disease. Although conditional expression of the NrasG12D/ + mutation in adult hematopoietic system is leukemogenic, its effects on embryonic development remain unclear. Here, we report that pan-embryonic expression of endogenous NrasG12D/ + by Mox2-Cre in mice caused embryonic lethality from embryonic day (E) 15.5 and developmental defects predominantly in the heart. At E13.5, NrasG12D/ + ; Mox2Cre/ + embryos displayed a moderate expansion of hematopoietic stem and progenitor cells without a significant impact on erythroid differentiation in the fetal liver. Importantly, the mutant embryos exhibited cardiac malformations resembling human congenital cardiac defects seen in NS patients, including ventricular septal defects, double outlet right ventricle, the hypertrabeculation/thin myocardium, and pulmonary valve stenosis. The mutant heart showed dysregulation of ERK, BMP, and Wnt pathways, crucial signaling pathways for cardiac development. Endothelial/endocardial-specific expression of NrasG12D/ + caused the cardiac morphological defects and embryonic lethality as observed in NrasG12D/ + ; Mox2Cre/ + mutants, but myocardial-specific expression of NrasG12D/ + did not. Thus, oncogenic NrasG12D mutation may not be compatible with embryonic survival.

Expression of NrasQ61R and MYC transgene in germinal center B cells induces a highly malignant multiple myeloma in mice.

NRAS Q61 mutations are prevalent in advanced/relapsed multiple myeloma (MM) and correlate with poor patient outcomes. Thus, we generated a novel MM model by conditionally activating expression of endogenous NrasQ61R and an MYC transgene in germinal center (GC) B cells (VQ mice). VQ mice developed a highly malignant MM characterized by a high proliferation index, hyperactivation of extracellular signal-regulated kinase and AKT signaling, impaired hematopoiesis, widespread extramedullary disease, bone lesions, kidney abnormalities, preserved programmed cell death protein 1 and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain immune-checkpoint pathways, and expression of human high-risk MM gene signatures. VQ MM mice recapitulate most of the biological and clinical features of human advanced/high-risk MM. These MM phenotypes are serially transplantable in syngeneic recipients. Two MM cell lines were also derived to facilitate future genetic manipulations. Combination therapies based on MEK inhibition significantly prolonged the survival of VQ mice with advanced-stage MM. Our study provides a strong rationale to develop MEK inhibition-based therapies for treating advanced/relapsed MM.

Downregulating Notch counteracts KrasG12D-induced ERK activation and oxidative phosphorylation in myeloproliferative neoplasm.

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia.

Unique dependence on Sos1 in Kras G12D -induced leukemogenesis.

We and others have previously shown that Kras G12D is a much more potent oncogene than oncogenic Nras in hematological malignancies. We attributed the strong leukemogenic activity of KrasG12D at least partially to its unique capability to hyperactivate wild-type (WT) Nras and Hras. Here, we report that Sos1, a guanine nucleotide exchange factor, is required to mediate this process. Sos1 is overexpressed in Kras G12D/+ cells, but not in Nras Q61R/+ and Nras G12D/+ cells. KrasG12D proteins form a complex with Sos1 in vivo. Sos1 deficiency attenuates hyperactivation of WT Nras, Hras, and the downstream ERK signaling in Kras G12D/+ cells. Thus, Sos1 deletion ameliorates oncogenic Kras-induced myeloproliferative neoplasm (MPN) phenotypes and prolongs the survival of Kras G12D/+ mice. In contrast, Sos1 is dispensable for hyperactivated granulocyte-macrophage colony-stimulating factor signaling in Nras Q61R/+ cells, and Sos1 -/- does not affect MPN phenotypes in Nras Q61R/+ mice. Moreover, the survival of Kras G12D/+ ; Sos1 -/- recipients is comparable to that of Kras G12D/+ recipients treated with combined MEK and JAK inhibitors. Our study suggests that targeting Sos1-oncogenic Kras interaction may improve the survival of cancer patients with KRAS mutations.

The mystery of oncogenic KRAS: Lessons from studying its wild-type counter part.

Using conditional knock-in mouse models, we and others have shown that despite the very high sequence identity between Nras and Kras proteins, oncogenic Kras displays a much stronger leukemogenic activity than oncogenic Nras in vivo. In this manuscript, we will summarize our recent work of characterizing wild-type Kras function in adult hematopoiesis and in oncogenic Kras-induced leukemogenesis. We attribute the strong leukemogenic activity of oncogenic Kras to 2 unique aspects of Kras signaling. First, Kras is required in mediating cell type- and cytokine-specific ERK1/2 signaling. Second, oncogenic Kras, but not oncogenic Nras, induces hyperactivation of wild-type Ras, which significantly enhances Ras signaling in vivo. We will also discuss a possible mechanism that mediates oncogenic Kras-evoked hyperactivation of wild-type Ras and a potential approach to down-regulate oncogenic Kras signaling.

Kras is Required for Adult Hematopoiesis.

Previous studies indicate that Kras is dispensable for fetal liver hematopoiesis, but its role in adult hematopoiesis remains unclear. Here, we generated a Kras conditional knockout allele to address this question. Deletion of Kras in adult bone marrow (BM) is mediated by Vav-Cre or inducible Mx1-Cre. We find that loss of Kras leads to greatly reduced thrombopoietin (TPO) signaling in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), while stem cell factor-evoked ERK1/2 activation is not affected. The compromised TPO signaling is associated with reduced long term- and intermediate-term HSC compartments and a bias toward myeloid differentiation in MPPs. Although granulocyte macrophage colony-stimulating factor (GM-CSF)-evoked ERK1/2 activation is only moderately decreased in Kras(-/-) myeloid progenitors, it is blunted in neutrophils and neutrophil survival is significantly reduced in vitro. At 9-12 months old, Kras conditional knockout mice develop profound hematopoietic defects, including splenomegaly, an expanded neutrophil compartment, and reduced B cell number. In a serial transplantation assay, the reconstitution potential of Kras(-/-) BM cells is greatly compromised, which is attributable to defects in the self-renewal of Kras(-/-) HSCs and defects in differentiated hematopoietic cells. Our results demonstrate that Kras is a major regulator of TPO and GM-CSF signaling in specific populations of hematopoietic cells and its function is required for adult hematopoiesis. Stem Cells 2016;34:1859-1871.

Kras Is Critical for B Cell Lymphopoiesis.

The three major Ras members, Kras, Hras, and Nras, are highly homologous and individual Ras genes can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological functions of this Ras family member. In this study, we generated and examined mice with hematopoietic-specific deletion of Kras and bone marrow (BM) chimeric mice with B cell-specific targeted deletion of Kras. Hematopoietic-specific deletion of Kras impaired early B cell development at the pre-B cell stage and late B cell maturation, resulting in the reduction of BM pre-, immature, and mature B cells and peripheral follicular, marginal zone, and B1 mature B cells. In contrast, Kras deficiency did not affect T cell development. Studies of BM chimeric mice with B cell-specific deletion of Kras demonstrated that Kras deficiency intrinsically impaired B cell development. Kras deficiency reduced BCR-induced B cell proliferation and survival. Furthermore, Kras deficiency specifically impaired pre-BCR- and BCR-induced activation of the Raf-1/MEK/ERK pathway in pre-B and mature B cells, respectively. Thus, Kras is the unique Ras family member that plays a critical role in early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway.

The oncoprotein HBXIP promotes glucose metabolism reprogramming via downregulating SCO2 and PDHA1 in breast cancer.

The glucose metabolism reprogramming is a hallmark of cancer. The oncoprotein hepatitis B X-interacting protein (HBXIP) functions in the development of breast cancer. In this study, we supposed that HBXIP might be involved in the glucose metabolism reprogramming in breast cancer. We showed that HBXIP led to increases in generation of intracellular glucose and lactate, as well as decreases in generation of reactive oxygen species. Expression of synthesis of cytochrome c oxidase 2 (SCO2) and pyruvate dehydrogenase alpha 1 (PDHA1), two factors of metabolic switch from oxidative phosphorylation to aerobic glycolysis, was suppressed by HBXIP. In addition, miR-183/182 and miR-96 directly inhibited the expression of SCO2 and PDHA1 through targeting their mRNA coding sequences (CDSs), respectively. Interestingly, HBXIP elevated the miR-183/96/182 cluster expression through hypoxia-inducible factor 1α (HIF1α). The stability of HIF1α was enhanced by HBXIP through disassociating interaction of von Hippel-Lindau protein (pVHL) with HIF1α. Moreover, miR-183 increased the levels of HIF1α protein through directly targeting CDS of VHL mRNA, forming a feedback loop of HIF1α/miR-183/pVHL/HIF1α. In function, HBXIP-elevated miR-183/96/182 cluster enhanced the glucose metabolism reprogramming in vitro. HBXIP-triggered glucose metabolism reprogramming promoted the growth of breast cancer in vivo. Thus, we conclude that the oncoprotein HBXIP enhances glucose metabolism reprogramming through suppressing SCO2 and PDHA1 in breast cancer.

A Prospective Randomized Study of Anterior Cruciate Ligament Reconstruction With Autograft, γ-Irradiated Allograft, and Hybrid Graft.

PURPOSE: To compare the results of patients who underwent anterior cruciate ligament (ACL) reconstruction with autograft, γ-irradiated allograft, or hybrid graft in a prospective randomized study with a minimum clinical follow-up period of 5 years. METHODS: In this prospective, randomized, comparative study, 102 patients with ACL tears underwent ACL reconstruction with autograft (gracilis and semitendinosus tendons), γ-irradiated allograft (tibialis anterior tendons), or hybrid graft (γ-irradiated tibialis anterior tendon allograft and semitendinosus tendon autograft). Laboratory testing of the erythrocyte sedimentation rate and C-reaction protein level were performed; clinical results were evaluated with the KT-1000 arthrometer (MEDmetric, San Diego, CA), Lachman test, Lysholm score, Tegner activity score, and International Knee Documentation Committee evaluation (both objective and subjective). RESULTS: There were 32 patients in the autograft group, 31 in the hybrid graft group, and 32 in the γ-irradiated allograft group at last follow-up. No differences were found among the 3 groups regarding patient demographic data and the duration from injury to operation (P > .05). The C-reaction protein and erythrocyte sedimentation rate values were statistically higher in the γ-irradiated allograft group than in the other 2 groups on the third, seventh, and fourteenth days (P < .05). No significant differences were found between the autograft and hybrid graft groups (P > .05). The KT-1000 examination showed more anterior laxity in the γ-irradiated allograft group than in the other 2 groups (P < .05). No significant differences in the Lachman test and pivot-shift test findings were found among the 3 groups (P > .05). The Lysholm score, Tegner activity score, and International Knee Documentation Committee evaluation (subjective and objective) showed no differences among the 3 groups (P > .05). CONCLUSIONS: Patients undergoing primary ACL reconstruction with hybrid graft or autograft had satisfactory and similar objective and subjective clinical results. On KT-1000 measurement of anteroposterior knee laxity, both the autograft and hybrid graft groups showed statistically significant differences compared with the γ-irradiated allograft group. LEVEL OF EVIDENCE: Level II, prospective comparative study.