Increased lactate in AML blasts upregulates TOX expression, leading to exhaustion of CD8+ cytolytic T cells.

Recently, the role of lactate as merely an end product of cancer cell metabolism has been reassessed. Lactate has been implicated in more biological processes than previously understood and drives tumor progression. Here, we demonstrated that the bone marrow lactate concentrations in acute myeloid leukemia (AML) patients were substantially higher than those in their healthy control counterparts. Moreover, AML blasts from bone marrow expressed significantly higher lactate dehydrogenase-A (LDHA) levels. Further studies revealed that LDHA expression was regulated through the HIF1α pathway. Elevated lactate levels were indicative of alterations in CD8+ T cell cytolytic phenotype and activity. An in vitro study showed that the lactate treatment group had significantly higher percentages of CD8+ TEM and CD8+ TEMRA cells as well as higher PD-1 expression in these cells than the control group. Lactate induced the loss of the effector function of CD8+ T cells by altering lytic granule exocytosis. T cell dysfunction is characterized by an increase in terminally differentiated phenotypes, sustained expression of PD-1, and accelerated decline of cytolytic competence. Moreover, the TOX gene was found to be correlated with lactate production and implicated in CD8+ T cell dysfunction. AML patients in complete remission after chemotherapy had markedly lower lactate concentrations, reduced CD8+ TEM and CD8+ TEMRA cells and PD-1 expression, and increased perforin and granzyme B. However, no difference was found in the relapsed patients. The study presented here has established lactate as a predictive biomarker for patient response to antitumor therapies and demonstrated that targeting this gene in AML patients could be a meaningful precision therapeutic strategy.

Inhibition of USP1 induces apoptosis via ID1/AKT pathway in B-cell acute lymphoblastic leukemia cells.

Deubiquitylating enzyme ubiquitin-specific protease 1 (USP1) has been reported to be aberrantly overexpressed in cancers, and it plays a critical role in regulating various cellular processes, such as cell proliferation, apoptosis, and cell differentiation. However, the role of USP1 in B-cell acute lymphoblastic leukemia (B-ALL) remains largely undefined. USP1 expression in 30 newly diagnosed B-ALL patients was detected by real-time PCR and western blot. We found that USP1 was generally upregulated in the bone marrow cells derived from B-ALL patients. Knockdown of USP1 by siRNA decreased B-ALL cell growth and induced apoptosis. Similarly, pharmacological inhibition of USP1 by SJB3-019A significantly repressed cell proliferation and triggered B-ALL cell apoptosis. Finally, we found that inhibition of USP1 downregulated the expression of ID1 and p-AKT, and upregulated ID1 expression could reverse the suppressive effects of USP1 inhibitor in B-ALL cells. Taken together, these results demonstrate that USP1 promote B-ALL progression at least partially via the ID1/AKT signaling pathway, and USP1 inhibitors might be promising therapeutic application for B-ALL.

C3a and C5a facilitates the metastasis of myeloma cells by activating Nrf2.

Multiple myeloma (MM) is still an incurable hematological malignancy, with even poorer prognosis in MM patients with distant invasion. The present study was designed to explore the effects of C3a and C5a on the migration, invasion, and adhesion of MM tumor cells and to investigate the underlying mechanisms. As a result, the levels of C3a and C5a in plasma of MM patients were significantly higher than those of healthy donors. Consistently, the expression of C3a and C5a receptors on myeloma cells of MM patients was also significantly higher than that on sorted plasma cells of normal donors. C3a and C5a have been confirmed to increase the migration, invasion and adhesion of MM cell lines by activating the MEK/ERK pathway and increasing the nuclear transfer of Nrf2 in vitro. Moreover, the MM cell line U266 with Nrf2 downregulation was incubated with C3a and C5a, followed by injection into the tail vein of NOD-SCID mice. We found that Nrf2 downregulation attenuated the migration of anaphylatoxin C3a and C5a to MM tumor cells in bone marrow, liver and lung in vivo. In conclusion, our results indicate that activation of the complement cascade in MM patients may contribute to the migration, invasion and adhesion of MM cells, and this type of tumor cells dissemination in MM is, at least partially, regulated by Nrf2. Thereby, complement suppression or Nrf2 downregulation might offer a novel therapeutic opportunity for MM.

Clonal expansion of bone marrow CD8+ T cells in acute myeloid leukemia patients at new diagnosis and after chemotherapy.

CD8+ T cells are crucial adaptive immune effectors and express receptors (T cell receptors, TCRs) that specifically recognize and eradicate tumor cells. The diversity of the TCR repertoire is generated by specialized genetic diversification mechanisms, which lead to an extremely variable TCR repertoire that is capable of recognizing a wide range of antigens. However, the variations in CD8+ TCR diversity and their clinical implications in acute myeloid leukemia (AML) patients remain unknown. CD8+ T cells were enriched from 10 healthy donors and 31 AML patients at diagnosis and after chemotherapy, and TCRß deep sequencing was performed to analyze CD8+ T cell clonal expansion and TCR repertoire diversity. Diminished TCR repertoire diversity and increased T cell clone expansion were noted in the bone marrow of AML patients. In relapsed patients, T cells were found to be more clonally expanded after chemotherapy than at new diagnosis. Moreover, significantly more expanded TCRß clonotypes were noted in CD8+ PD-1+ T cells than in CD8+ PD-1- T cells regardless of the time of examination. Our systematic T cell repertoire analysis may help better characterize CD8+ T cells before and after chemotherapy in AML, which may provide insights into therapeutic strategies for hematological malignancies.

4SC-202 induces apoptosis in myelodysplastic syndromes and the underlying mechanism.

Epigenetic modifications play crucial roles in regulating the self-renewal and differentiation of hematopoiesis. 4SC-202, a novel inhibitor of histone lysine-specific demethylase 1 (LSD1) and class I histone deacetylases (HDACs), is a potential therapeutic agent to treat myelodysplastic syndrome (MDS). However, it remains unclarified of the mechanism of 4SC-202. In the study, we found that 4SC-202 treatment could inhibit cell viability, induce apoptosis and cause G2/M cell cycle arrest in MDS cell line SKM-1. Heme oxygenase-1 (HO-1) was correlated with disease progression and chemotherapy resistance. Here, we reported that 4SC-202 could down-regulate the expression of HO-1, and up-regulation of HO-1 could significantly attenuate the 4SC-202-induced apoptosis in SKM-1 cells. In addition, the activation of NF-κB pathway was suppressed by 4SC-202, while up-regulation of HO-1 significantly weakened the 4SC-202-induced suppression of the NF-κB pathway, thereby attenuating the efficacy of 4SC-202. However, down-regulation of HO-1 enhanced the sensitivity of 4SC-202 against SKM-1 cells. Moreover, SKM-1 cells were transfected with HO-1 overexpression lentivirus, subsequently injected into the tail vein of NOD/SCID mice, followed by administration of 4SC-202 in mice. As a result, up-regulation HO-1 could partially attenuate 4SC-202-suppressed MDS cells growth in NOD/SCID mice. In conclusion, 4SC-202 could induce apoptosis via the NF-κB pathway, and our present finding may provide a novel therapeutic strategy for MDS.

Heme oxygenase-1 inhibition mediates Gas6 to enhance bortezomib-sensitivity in multiple myeloma via ERK/STAT3 axis.

Chemoresistance is still a critical challenge for efficient treatment of multiple myeloma (MM) during the bortezomib-based chemotherapy. Recent studies have suggested that heme oxygenase-1 (HO-1) is involved in apoptosis, proliferation and chemoresistance in cancer cells. Here we aim to investigate the role and mechanism of HO-1 in bortezomib-sensitivity to myeloma cells. In the study population, we found that HO-1 was highly expressed in CD138+ primary myeloma cells, which was positively associated with Gas6 expression and Gas6 plasma levels in MM patients. Downregulation of HO-1 using pharmacological inhibitor ZnPPIX or siRNA knockdown significantly enhanced myeloma cell sensitivity to bortezomib in human primary CD138+ cells, U266 and RPMI8226 cell lines. Mechanistically, HO-1 regulated Gas6 production via ERK/STAT3 axis. Combination with HO-1 inhibition increased bortezomib-induced apoptosis and antiproliferative effects via suppressing Gas6 production. These findings suggest that combination of bortezomib and HO-1 inhibitor may serve as a promising therapeutic target against bortezomib-resistant MM.

Fenretinide-induced Apoptosis of Acute Myeloid Leukemia Cells via NR4A1 Translocation into Mitochondria and Bcl-2 Transformation.

OBJECTIVE: Fenretinide is reported to induce NR4A1-associated apoptosis in several types of cancer cells. However, it remains unclear about its specific role and the underlying mechanism in acute myeloid leukemia (AML). Therefore, this study aimed to explore the role and mechanism of fenretinide-induced apoptosis in AML. METHOD: Firstly, the NR4A1 mRNA level in the newly diagnosed AML patients was measured, then AML cells were treated with fenretinide at various time points and doses, and cell viability was investigated by using the cell-counting kit-8 (CCK-8) assay. Additionally, apoptosis and cell cycles were analyzed by using flow cytometry. Moreover, siNR4A1 was utilized to knockdown NR4A1 expression, and leptomycin B (LMB) was adopted to inhibit the nuclear export; afterwards, the apoptosis rate and expression of apoptotic proteins in AML cells were detected. In addition, the expression levels of NR4A1 in the nuclei and mitochondria of fenretinide-treated AML cells were also measured. Meanwhile, the interaction between NR4A1 and Bcl-2, as well as the Bcl-2 transformation, was also examined. The anti-leukemic effect of fenretinide on NOD/SCID mice was also determined through subcutaneous injection of HL-60 cells. RESULTS: NR4A1 expression in AML patients was markedly down-regulated compared with that in normal donors. Fenretinide induced the expression of NR4A1 and mitochondria-mediated apoptotic pathway-associated proteins in a time- and concentration-dependent manner. Importantly, both siNR4A1 alone or the combination of fenretinide with LMB could attenuate the fenretinide-induced apoptosis and expression of apoptotic proteins. Under the action of fenretinide, the NR4A1 protein expression was down-regulated in nuclear extracts whereas up-regulated in mitochondrial extracts. At the same time, fenretinide promoted NR4A1 translocation from nuclei into mitochondria, and enhanced the interaction between NR4A1 and Bcl-2, thereby exposing the BH3 domain of Bcl-2 to exert the anti-apoptotic effect. Moreover, fenretinide also exhibited an anti-leukemic effect and induced NR4A1 expression in the AML mouse model. CONCLUSIONS: Fenretinide exerts an obvious effect on AML cells both in vitro and in vivo. Besides, the NR4A1-mediated signaling pathway is highly involved in the fenretinide-induced apoptosis of AML cells.

Histone deacetylase inhibitor LMK-235-mediated HO-1 expression induces apoptosis in multiple myeloma cells via the JNK/AP-1 signaling pathway.

AIMS: Histone deacetylase inhibitors (HDACis) are promising anticancer drugs that open new areas of epigenetic drug discovery. Multiple myeloma (MM) is a malignant tumor of the blood system that is difficult to cure and often relapses. Here, we investigated the in vitro effects of a novel HDACi, LMK-235, on MM cells, and explored the underlying mechanisms. MAIN METHODS: Real-time PCR and western blot were used to measure the expression of HDAC4 and HO-1 in MM cells treated with LMK-235. si-RNA was used to transfect MM cells. Hemin or ZnPP was combined to regulate heme oxygenase-1 (HO-1), and a pathway inhibitor was added to measure changes in the JNK/AP-1 signaling pathway. Apoptosis and proliferation were assessed by flow cytometry and CCK-8 assay, respectively. KEY FINDINGS: We found that LMK-235, a selective inhibitor of class IIA HDAC4/5, induced apoptosis of MM cells by downregulating HO-1 that is closely related to HDAC4. LMK-235 increased phosphorylation of JNK and c -Jun in MM cells. Downregulation of HO-1 expression in combination with LMK-235 expression further activated phosphorylation of JNK and c-Jun and induced apoptosis in MM cells. When the JNK inhibitor SP600125 was used in combination, the apoptosis phenomenon was reversed. However, when HO-1 was upregulated, LMK-235-mediated phosphorylation of JNK and c-Jun was inhibited, and apoptosis of MM cells began to decrease. SIGNIFICANCE: These data suggest that LMK-235 has potent anti-myeloma activity through regulation of HO-1-induced apoptosis via the JNK/AP-1 pathway. This provides a new concept for the treatment of multiple myeloma.

PIM inhibitor SMI-4a induces cell apoptosis in B-cell acute lymphocytic leukemia cells via the HO-1-mediated JAK2/STAT3 pathway.

OBJECTIVES: The serine/threonine PIM protein kinases are critical regulators of tumorigenesis in multiple cancers. However, whether PIMs are potential therapeutic targets for treating B-cell acute lymphocytic leukemia (B-ALL) remains unclear. Therefore, here, PIM expression was detected in B-ALL patients and the effects of SMI-4a, a pan-PIM small molecule inhibitor, were investigated in B-ALL cells. METHODS: PIM1 and PIM2 expression in 26 newly diagnosed B-ALL cases was detected by real-time PCR and Western blot. B-ALL cells were treated with varied SMI-4a doses and the viability of treated cells was investigated using a cell-counting kit-8 (CCK-8) assay. Apoptosis and cell cycles were analyzed by flow cytometry. Western blot analysis was then used to explore the expression of apoptosis-related proteins and the JAK2/STAT3 pathway. RESULTS: PIM1 and 2 were overexpressed in B-ALL patients with high HO-1 level. SMI-4a induced decreases in PIMs and HO-1 expressions and inhibited B-ALL cell viability. Treatment with SMI-4a induced apoptosis by downregulating Bcl-2, upregulating Bax and other antiapoptotic proteins, and decreasing protein levels of p-JAK2 and p-STAT3. In addition, upregulation of HO-1 alleviated decrease in p-JAK2 and p-STAT3 expression, reduced SMI-4a-induced apoptosis of B-ALL cells, and influenced B-ALL cell survival. CONCLUSIONS: PIMs were highly expressed in B-ALL patients. SMI-4a inhibited B-ALL proliferation and induced apoptosis via the HO-1-mediated JAK2/STAT3 pathway. SMI-4a might be applicable for treatment of B-ALL cells.

MicroRNA­143 increases cell apoptosis in myelodysplastic syndrome through the Fas/FasL pathway both in vitro and in vivo.

Whilst the role of microRNA­143 (miR­143) in myelodysplastic syndrome (MDS) remains unclear, abnormally expressed microRNA­143 has been detected in many types of cancer tissues. In this study, we describe a cohort study for the verification of miR­143 expression, as well as the investigation of the molecular mechanisms of miR­143 in MDS/acute myeloid leukaemia (AML). In a series of experiments, miR­143 recombinant lentiviral vectors transformed into SKM­1 cells were either overexpressed or knocked down, and the results illustrated that the overexpression of miR­143 inhibited SKM­1 cell growth, arrested the SKM­1 cells in the G0/G1 phase, interfered with cell proliferation and induced cell apoptosis via the Fas/FasL pathway. Conversely, miR­143 knockdown induced a decrease in the apoptosis and promoted the proliferation of SKM­1 cells. Moreover, miR­143 was shown to suppress MLLT3/AF9 expression by binding to its 3'­UTR. Taken together, the findings of this study indicate that miR­143 may be a critical regulator of MDS/AML cell carcinogenesis, acting as a potent antitumour molecular target for the diagnosis or treatment of cancers associated with the abnormal expression of MLLT3/AF9, hence facilitating the development of potential therapeutics against MDS/AML.

Effect of BCLAF1 on HDAC inhibitor LMK-235-mediated apoptosis of diffuse large B cell lymphoma cells and its mechanism.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of adult lymphoma. It is a group of malignant tumors with a large number of clinical manifestations and prognoses. Therefore, it is necessary to explore its unknown potential therapeutic targets. Histone deacetylase inhibitor (HDACi) is a novel drug for the treatment of DLBCL, however pan-HDACis cannot be ignored because of their clinical efficacy. By contrast, specific HDACi is well-tolerated, and LMK-235 is a novel HDACi that is a specific inhibitor of HDAC4 and HDAC5. In this study, we investigated the up-regulation of BCLAF1 through NF-κB signaling pathways in LMK-235, mediating the apoptosis of two diffuse large B-cell lymphoma cell lines, OCI-LY10 and OCI-LY3. Further studies showed that BCLAF1 expression was increased in DLBCL cells after treatment with the NF-κB inhibitor Bay11-7082. The combination of Bay11-7082 and siRNA si-HDAC4 significantly increased BCLAF1 expression and further increased apoptosis. These results indicate that BCLAF1 plays an important role in LMK-235-mediated apoptosis and may be a potential target for the treatment of diffuse large B-cell lymphoma.

Crucial role of HO-1/IRF4-dependent apoptosis induced by panobinostat and lenalidomide in multiple myeloma.

Inhibition of histone deacetylase (HDAC) is a promising therapeutic strategy for various hematologic cancers. Panobinostat has been approved for treating patients with multiple myeloma (MM) by the FDA. Since the mechanism for the resistance of panobinostat to MM remains elusive, we aimed to clarify this mechanism and the synergism of panobinostat with lenalidomide. The mRNA and protein of transcription factor IRF4 were overexpressed in CD138+ mononuclear cells from MM patients compared with in those from healthy donors. Given that direct IRF4 inhibitors are clinically unavailable, we intended to explore the mechanism by which IRF4 expression was regulated in MM. Heme oxygenase-1 (HO-1) promotes the growth and drug resistance of various malignant tumors, and its expression is positively correlated with IRF4 mRNA and protein expression levels. Herein, panobinostat induced acetylation of histone H3K9 and activation of caspase-3 in MM cells, being inversely correlated with the reduction of HO-1/IRF4/MYC protein levels. Adding Z-DEVD-FMK, a caspase-3 inhibitor, abolished the HO-1/IRF4 reduction by panobinostat alone or in combination with lenalidomide, suggesting that caspase-3-mediated HO-1/IRF4/MYC degradation occurred. Given that lenalidomide stabilized cereblon and facilitated IRF4 degradation in MM cells, we combined it with LBH589, an HDAC inhibitor. LBH589 and lenalidomide exerted synergistic effects, and LBH589 reversed the efficacy of lenalidomide on the resistance of CD138+ primary MM cells, in part due to simultaneous suppression of HO-1, IRF4 and MYC. The results provide an eligible therapeutic strategy for targeting MM depending on the IRF4 network and clinical testing of this drug combination in MM patients.

miR-378 inhibits cell growth and enhances apoptosis in human myelodysplastic syndromes.

miR-378 has been proven to inhibit cell growth, migration and invasion in different types of cancers. In this study, we found that miR-378 was commonly downregulated in the bone marrow cells obtained from myelodysplastic syndrome (MDS) patients. We further investigated the role of miR-378 in the proliferation and apoptosis of SKM-1 cells, an acute myeloid leukemia cell line established in the leukemic phase during the progression of MDS to AML (MDS/AML). Results indicated that overexpression of miR-378 in SKM-1 cells interfered with proliferation via inducing apoptosis and G0/G1-phase cell cycle arrest, and suppressive effect of miR-378 on MDS/AML cells may be mediated partly through Bcl-w and CDC40. Moreover, apoptosis induced by miR-378 correlated with increased expression of Bax and activation of caspase-3, -8 and -9. Taken together, our data support a critical role for miR-378 in the pathogenesis of MDS and provide a novel therapeutic target in this complex disease.

Deregulated microRNA expression and its pathogenetic implications for myelodysplastic syndromes.

OBJECTIVE: Myelodysplastic syndromes (MDS) include a heterogeneous group of clonal hematological stem cell disorders characterized by ineffective hematopoiesis, cytopenias. MicroRNAs (miRNAs) are short non-coding RNA molecules that repress gene expression at the post-transcriptional level. In this review, we summarize advanced investigations that underscore deregulated miRNA expression in MDS, and discuss the implications of miRNAs in the molecular pathogenesis of MDS. METHODS: Relevant English-language literatures were searched and retrieved from PubMed using the terms MDS and miRNAs. RESULTS: The majority of studies have focused on profiling miRNA expression in MDS, only a small number of studies have investigated the exact pathogenic role of miRNAs in MDS. DISCUSSION: In the hematopoietic system, miRNAs are critical regulators of the differentiation of hematopoietic stem/progenitor cells. Thus, it is not surprising that dysregulation of miRNAs can lead to hematopoietic stem cell anomalies and further cause MDS. Deregulated miRNA expression has been identified in MDS, and it contributes to the pathogenesis and progression of MDS. Chromosomal aberrations, hypermethylation of miRNA promoters, and mutations of miRNA genes may lead to dysregulation of miRNA in MDS. However, the complex regulatory networks between miRNAs and their potential target genes in MDS still need to be explored in further studies. CONCLUSIONS: Although the function of miRNAs is not fully understood, these small non-coding RNAs represent novel pathogenetic and clinical implications in MDS. The studies of miRNAs may guide us towards a better understanding of this disease and shed light on the development of new therapeutic strategies.

Fucoidan inhibits proliferation of the SKM-1 acute myeloid leukaemia cell line via the activation of apoptotic pathways and production of reactive oxygen species.

Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid disorders characterized by peripheral blood cytopenias and a high risk of progression to acute myeloid leukaemia (AML). Fucoidan, a complex sulphated polysaccharide isolated from the cell wall of brown seaweeds, has recently attracted attention for its multiple biological activities and its potential as a novel candidate for cancer therapy. In the present study, the anti­cancer activity of fucoidan was investigated in the MDS/AML cell line SKM­1. Fucoidan inhibited proliferation, induced apoptosis and caused G1-phase arrest of the cell cycle in SKM­1 cells as determined by a cell counting kit 8 assay and flow cytometry. Furthermore, reverse transcription quantitative polymerase chain reaction and western blot analyses indicated that treatment with fucoidan (100 µg/ml for 48 h) activated Fas and caspase­8 in SKM­1 cells, which are critical for the extrinsic apoptotic pathway; furthermore, caspase­9 was activated via decreases in phosphoinositide-3 kinase/Akt signaling as indicated by reduced levels of phosphorylated Akt, suggesting the involvement of the intrinsic apoptotic pathway. In addition, fucoidan treatment of SKM­1 cells resulted in the generation of reactive oxygen species (ROS) as determined by staining with dichloro-dihydro-fluorescein diacetate. These results suggested that the mechanisms of the anti­cancer effects of fucoidan in SKM­1 are closely associated with cell cycle arrest and apoptotic cell death, which partly attributed to the activation of apoptotic pathways and accumulation of intracellular ROS. Our results demonstrated that Fucoidan inhibits proliferation and induces the apoptosis of SKM­1 cells, which provides substantial therapeutic potential for MDS treatment.

Gene expression profiling in myelodysplastic syndrome after SPARC overexpression associated with Ara-C.

Secreted protein acidic and rich in cysteine (SPARC) is involved in many biological processes, including erythropoiesis and cell proliferation. However, the role of SPARC in myelodysplastic syndrome (MDS) remains to be elucidated. Pyrimidine analogue cytosine arabinoside (Ara-C) is among the most effective agents used in the treatment of acute leukemia. The aim of the present study was to determine whether the chemotherapeutic activity of Ara-C was enhanced by the overexpression of SPARC. DNA microarray technology and RNA sequencing were employed to examine differential gene expression in the apoptosis signaling pathway after gene change occurred in cells following drug treatment. The results showed that upregulation of the expression of SPARC induced SKM-1 cell death and inhibited proliferation. Additionally, the apoptotic rate of SPARC overexpression combined with Ara-C increased significantly. Transcription factors CPBP and ZNF333 regulated the 69 genes and long non-coding RNA (lncRNA). Moreover, the mRNA and protein expression of apoptosis-related genes in the DNA microarray results were increased. These results suggest that SPARC expression changes with Ara-C, revealing a possible application in the treatment of MDS.