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Our previous data showed that young female multiple myeloma (MM) patients had a low frequency of osteolytic lesions. Based on this clinical observation, we found that estrogen cell signaling played a regulatory role in MM bone disease (MMBD), and the estrogen-responsive gene microtubule-associated serine/threonine kinase family member 4 (MAST4) was a critical factor. The presence of estrogen in cell cultures promoted MAST4 expression in MM cells, while knocking down estrogen receptor 1 (ESR1) inhibited MAST4 expression. Chromatin immunoprecipitation assay suggested a binding site of ESR1 on the MAST4 promoter. Bisphosphonates, such as zoledronic acid (ZOL), which was widely used in MMBD control, could stimulate MAST4 expression in MM cells by promoting ESR1 expression. MAST4 interacted with phosphatase and tensin homolog (PTEN), therefore regulating the PI3K-Akt-mTOR pathway and the expression of downstream cytokines, such as CCL2/3/4. MAST4 knockdown (MAST4-KD) or ESR1 knockdown (ESR1-KD) MM cells had repressed PTEN activity, elevated PI3K-Akt-mTOR activity, and increased CCL2/3/4 expressions. Coculture of MAST4-KD or ESR1-KD MM cells with pre-osteoclasts (pre-OCs) stimulated OC formation in vitro, whereas neutralizing antibodies of CCL2/3/4 attenuated such stimulation. In mouse models, mice inoculated with MAST4-KD or ESR1-KD MM cells had severer MMBD than control knockdown (CTR-KD). The correlations between MAST4 and ESR1 expressions in MMBD, as well as related cell signaling pathways, were confirmed in analyses using gene expression profiles (GEPs) of patients' MM cells. The negative correlation of MAST4 expression and occurrence of MMBD was further validated by patients' immunohistochemical tissue array. Overall, our data suggested that estrogen cell signaling negatively regulated MMBD through MAST4. © 2022 American Society for Bone and Mineral Research (ASBMR).

Exosomal microRNAs (miRNAs) are potential biomarkers for a variety of tumors, but have not yet been studied in diffuse large B-cell lymphoma (DLBCL). Here, we investigated the use of exosomal miRNAs in DLBCL diagnosis and prognosis. A total of 256 individuals, including 133 DLBCL patients, 94 healthy controls (HCs), and 29 non-DLBCL concurrent controls (CCs), were enrolled. Exosomal miRNAs were profiled in the screening stage using microarray analysis, and miRNA candidates were confirmed in training, testing, and external testing stages using qRT-PCR. Follow-up information on the DLBCL patients was collected, and miRNAs were used to develop diagnostic and prognostic models for these patients. Five exosomal miRNAs (miR-379-5p, miR-135a-3p, miR-4476, miR-483-3p, and miR-451a) were differentially expressed between DLBCL patients and HCs with areas under the receiver operating characteristic curve (AUC) of 0.86, 0.90, and 0.86 for the training, testing, and external testing stages, respectively. Four exosomal miRNAs (miR-379-5p, miR-135a-3p, miR-4476, and miR-451a) were differentially expressed between patients with DLBCL and CCs, with an AUC of 0.78. One miRNA (miR-451a) was significantly associated with both progression-free survival (PFS) and overall survival (OS) of DLBCL patients, R analysis indicated the combination of miR-451a with international prognostic index was a better predictor of PFS and OS for these patients. Our study suggests that subsets of circulating exosomal miRNAs can be useful noninvasive biomarkers for the diagnosis of DLBCL and that the use of circulating exosomal miRNAs improves the identification of patients with newly diagnosed DLBCL with poor outcomes.

Multiple myeloma, a plasma cell malignancy in the bone marrow, remains largely incurable with currently available therapeutics. In this study, we discovered that the activated leukocyte cell adhesion molecule (ALCAM) interacted with epidermal growth factor receptor (EGFR), and regulated myelomagenesis. ALCAM was a negative regulator of myeloma clonogenicity. ALCAM expression was positively correlated with patients’ survival. ALCAM-knockdown myeloma cells displayed enhanced colony formation in the presence of bone marrow stromal cells (BMSCs). BMSCs supported myeloma colony formation by secreted epidermal growth factor (EGF), which bound with its receptor (EGFR) on myeloma cells and activated Mek/Erk cell signaling, PI3K/Akt cell signaling, and hedgehog pathway. ALCAM could also bind with EGFR, block EGF from binding to EGFR, and abolish EGFR-initiated cell signaling. Hence, our study identifies ALCAM as a novel negative regulator of myeloma pathogenesis.

The aim of this study was to investigate the effect of low-dose ruxolitinib (daily dose ≤ 10 mg) for the treatment of myelofibrosis (MF). A retrospective analysis was performed on a total of 88 patients with myeloproliferative neoplasm-associated MF (MPN-MF) who were diagnosed and treated in West China Hospital, Sichuan University, China. A total of 44 MPN-MF patients received a low dose of ruxolitinib (daily dose ≤ 10 mg), while another 44 patients received 10-25 mg twice daily. Low-dose ruxolitinib treatment resulted in slow, but gradual spleen response. Compared with baseline, the mean changes in palpable spleen length in the low- and high-dose groups were -26.9 and -49.0% after 12 weeks of treatment, respectively, and -46.7 and -64.1% after 48 weeks of treatment, respectively. In the low dose group, the median myeloproliferative neoplasm symptom assessment form (MPN-SAF) total symptom score (TSS) decreased by 37.8 and 35.9% at the 12 weeks and 48 weeks after treatment, respectively. No statistical difference was observed in MPN-SAF TSS among different dose groups. After 48 weeks of treatment, bone marrow (BM) fibrosis improved in 43.3% (13/30) of evaluated patients and was stable in 56.7% (17/30) patients. In the low-dose treated group, BM fibrosis improved in 50% patients and was stable in remaining 50%. Low-dose ruxolitinib is effective in treating MF.

This study aimed to investigate compassion satisfaction (CS) and compassion fatigue (CF) in haematology nurses and their associated factors.A cross-sectional survey.The survey was conducted on 336 haematology nurses from 21 hospitals in Sichuan, China. The CS and CF were assessed by the Professional Quality of Life Scale version 5. The CF was determined by burnout and secondary traumatic stress.Haematology nurses in China had moderate levels of CS and moderate-to-low CF. Better nursing competence of teaching/consultation and communication/coordination and the percentage of critically ill patients60% predicted higher CS. The permanent nurse, better nursing competence of communication/coordination and specialized clinical practice predicted less burnout, while working40 hr per week or more nurse-patient conflict events predicted more burnout. In addition, working40 hr per week, more nurse-patient conflict events and having the need of psychological support predicted higher secondary traumatic stress.

Drug-resistance is a major problem preventing a cure in patients with multiple myeloma (MM). Previously, we demonstrated that activated-leukocyte-cell-adhesion-molecule (ALCAM) is a prognostic factor in MM and inhibits EGF/EGFR-initiated MM clonogenicity. In this study, we further showed that the ALCAM-EGF/EGFR axis regulated the MM side population (SP)-mediated drug-resistance. ALCAM-knockdown MM cells displayed an enhanced ratio of SP cells in the presence of bone marrow stromal cells (BMSCs) or with the supplement of recombinant EGF. SP MM cells were resistant to chemotherapeutics melphalan or bortezomib. Drug treatment stimulated SP-genesis. Mechanistically, EGFR, primed with EGF, activated the hedgehog pathway and promoted the SP ratio; meanwhile, ALCAM inhibited EGFR downstream pro-MM cell signaling. Further, SP MM cells exhibited an increased number of mitochondria compared to the main population. Interference of the mitochondria function strongly inhibited SP-genesis. Animal studies showed that combination therapy with both an anti-MM agent and EGFR inhibitor gefitinib achieved prolonged MM-bearing mice survival. Hence, our work identifies ALCAM as a novel negative regulator of MM drug-resistance, and EGFR inhibitors may be used to improve MM therapeutic efficacy.

Appropriate biomarkers may help distinguish the biological behavior of different types of lymphoma and their response to traditional chemotherapy. Extranodal natural killer/T-cell lymphoma (ENKTL) and diffuse large B-cell lymphoma (DLBCL) belong to different subtypes of non-Hodgkin's lymphoma, the biological behavior and prognosis of them are very different, programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) have been investigated in these two types of diseases. However, few studies addressed the difference of PD-1/PD-L1 levels between ENKTL and DLBCL, in order to find out the difference and related clinical application value, the clinical data and tumor tissue paraffin sections of 24 newly diagnosed ENKTL patients and 42 newly diagnosed diffuse large B-cell lymphoma (DLBCL) were collected. PD-1/PD-L1 levels in tumor tissues were detected by immunohistochemical staining. The relationship between the PD-1/PD-L1 levels and clinical data of patients with ENKTL patients was analyzed. Both patient groups showed PD-1 level in tumor tissue of ENKTL patients was significantly lower than that of DLBCL patients (P

Multiple myeloma (MM) is the second most common hematological malignancy. It is characterized by abnormal transformation and uncontrolled clonal proliferation of malignant plasma cells in the bone marrow (BM), which can destroy bone structure and inhibit hematopoiesis. Although there are new therapeutic methods, they are not curative, mainly because it is difficult to deliver an effective amount of drug to BM, leading to a failure to eradicate MM cells inside the BM. BM homing is an important and unique characteristic of MM cells and it is mainly affected by surface molecules on the tumor cell membrane. Inspired by this mechanism, an MM-mimicking nanocarrier is developed by coating bortezomib (BTZ)-loaded poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCEC) nanoparticles with the MM cell membrane. The MM-mimicking nanoparticles can enter the BM based on BM homing as a "Trojan horse" and target the tumor cells through homologous targeting. In this way, drug availability at the myeloma site is enhanced so as to inhibit MM growth. In addition, these MM-mimicking nanoparticles can escape phagocytosis by the MPS and have a long circulation effect. The in vivo therapeutic results demonstrate an excellent treatment efficacy for MM. Accordingly, this strategy may be a promising platform for the treatment of MM.

The proteasome inhibitors (PIs) bortezomib and carfilzomib, which target proteasome 20S subunit beta 5 (PSMB5) in cells, are widely used in multiple myeloma (MM) treatment. In this study, we demonstrated the role of interferon-stimulated 20 kDa exonuclease-like 2 (ISG20L2) in MM PI resistance. Gain- and loss-of-function studies showed that ISG20L2 suppressed MM cell sensitivity to PIs in vitro and in vivo. Patients with ISG20L2lo MM had a better response to PIs and a longer overall survival than patients with ISG20L2hi MM. Biotinylated bortezomib pull-down assays showed that ISG20L2 competed with PSMB5 in binding to bortezomib. The surface plasmon resonance assay confirmed the direct binding of bortezomib to ISG20L2. In ISG20L2hi MM cells, ISG20L2 attenuated the binding of bortezomib to PSMB5, resulting in lower inhibition of proteasome activity and therefore less bortezomib-induced cell death. Overall, we identified a potentially novel mechanism by which ISG20L2 conferred bortezomib resistance on MM. The expression of ISG20L2 correlated with MM PI responses and patient treatment outcomes.

Multiple myeloma (MM) is an aggressive malignancy characterized by terminally differentiated plasma cells accumulation in the bone marrow (BM). MM BM exhibits elevated MΦs (macrophages) numbers relative to healthy BM. Current evidence indicates that MM-MΦs (MM-associated macrophages) have pro-myeloma functions, and BM MM-MΦs numbers negatively correlate with patient survival. Here, we found that BMI1, a polycomb-group protein, modulates the pro-myeloma functions of MM-MΦs, which expressed higher BMI1 levels relative to normal MΦs. In the MM tumor microenvironment, hedgehog signaling in MΦs was activated by MM-derived sonic hedgehog, and BMI1 transcription subsequently activated by c-Myc. Relative to wild-type MM-MΦs, BMI1-KO (BMI1 knockout) MM-MΦs from BM cells of BMI1-KO mice exhibited reduced proliferation and suppressed expression of angiogenic factors. Additionally, BMI1-KO MM-MΦs lost their ability to protect MM cells from chemotherapy-induced cell death. In vivo analysis showed that relative to wild-type MM-MΦs, BMI1-KO MM-MΦs lost their pro-myeloma effects. Together, our data show that BMI1 mediates the pro-myeloma functions of MM-MΦs.

Purpose: The aim of this study was to investigate the symptom clusters and associated clinical factors in ambulatory multiple myeloma patients undergoing medication therapy. We also aim to determine the correlations between symptom clusters and the patients’ quality of life. Methods: A total of 174 multiple myeloma patients hospitalized in the hematology day unit were included in this study. A cross-sectional survey aimed to examine the symptoms and quality of life was conducted. The symptoms were assessed by the Chinese version of Condensed Memorial Symptom Assessment Scale. The quality of life was measured with the Functional Assessment of Cancer Therapy-General. Principal component analysis was used to identify symptom clusters. Independent-Samples t test and Chi-square test were used for comparisons between groups. Spearman’s Rank Correlation Analysis was used to identify correlations.Results: We identified three symptom clusters in multiple myeloma patients: psychological, pain-dry mouth-difficulty sleep, and fatigue symptom cluster. For each symptom cluster, the patients could be categorized in severe-symptom group or mild-symptom group based on the distress of the symptoms. The patients in each group exhibited differential demographic and clinical features. The distress of each symptom cluster was adversely correlated with patients’ quality of life.Conclusions: The ambulatory multiple myeloma patients undergoing medication therapy experience multiple symptoms, which can be categorized into three symptom clusters. The distress of each symptom cluster was associated with patients’ demographic and clinical characteristics. The presence and distress of these symptom clusters have adverse impact on patient’s quality of life.

Acute myeloid leukemia (AML) with FLT3 internal tandem duplication (FLT3-ITD) has a dismal prognosis. FLT3 inhibitors have been developed to treat patients with FLT3-ITD AML; however, when used alone, their efficacy is insufficient. FLT3 inhibitors combined with chemotherapy may be a promising treatment for FLT3-ITD AML. Homoharringtonine (HHT) is a classical anti-leukaemia drug with high sensitivity to FLT3-ITD AML cells. Here, we showed that HHT synergizes with a selective next-generation FLT3 inhibitor, quizartinib, to inhibit cell growth/viability and induce cell-cycle arrest and apoptosis in FLT3-ITD AML cells in vitro, significantly inhibit acute myeloid leukemia progression in vivo, and substantially prolong survival of mice-bearing human FLT3-ITD AML. Mechanistically, HHT and quizartinib cooperatively inhibit FLT3-AKT and its downstream targets GSK3β, c-Myc, and cyclin D1, cooperatively up-regulate the pro-apoptosis proteins Bim and Bax, and down-regulate the anti-apoptosis protein Mcl1. Most strikingly, HHT and quizartinib cooperatively reduce the numbers of side-population (SP) and aldehyde dehydrogenase (ALDH)-positive cells, which reportedly are rich in LSCs. In conclusion, HHT combined with quizartinib may be a promising treatment strategy for patients with FLT3-ITD AML.

Aims To examine the feasibility and efficacy of self-help relaxation exercises in alleviating symptom distress in adult patients with acute leukemia (AL). Methods A pilot randomized controlled trial was used. Thirty adult patients with AL who were hospitalized in a teaching hospital were enrolled and randomly divided into a wait-list control group or an intervention group. The intervention group received self-help relaxation exercise twice per day for 4 weeks. The feasibility indicators, patients’ symptom distress were assessed by a blinded data collector. Results Twenty-nine patients completed the study. The recruitment rate, retention rate, and adherence rate was 65.2%, 93.3%, and 98.2%, respectively. The intervention group had a significantly decreased distress score for pain symptoms (F1, 27 = 6.594, P = .016, the partial η2 = 0.20, 90% confidence interval = 0.02-0.39). Conclusions Self-help relaxation exercises were feasible for the AL patients and significantly reduced their pain symptoms. Minor revision of the protocol for future definitive trials is needed.

Mantle cell lymphoma (MCL) is a relatively rare subtype of non-Hodgkin’s lymphoma. To identify molecular biomarkers in MCL, we performed immunohistochemistry tissue arrays using biopsies from 64 MCL patients diagnosed in West China Hospital from 2012 to 2016. TP53 mutation status in those patients was also examined by sequencing. The sequencing results showed TP53 mutations were highly heterogeneous in MCL. We identified four novel TP53 mutations in MCL: P151R, G199R, V218E, and G325R. The MCL patients with TP53 mutations had inferior progression-free survival (PFS, p = 0.002) and overall survival (OS, p = 0.011). Tissue array results showed the expression of p53, Sox11, or Pax5 alone did not correlate with the patient PFS and OS. However, the MCL patients with triple-positive expression of p53/Sox11/Pax5 had inferior PFS (p = 0.008) and OS (p = 0.002). Such risk stratification was independent to the mantle cell lymphoma international prognostic index (MIPI), Ki-67 value, and TP53 mutation status of the patients. The triple-positive patients might represent a subtype of high-risk MCL. Our findings might indicate a novel way to stratify MCL and predict patients’ prognosis.

Background Adult acute leukemia (AL) patients who receive chemotherapy usually experience multiple symptoms during the treatment course. The symptom clusters (SCs) as well as subsets of concurrent symptoms in AL patients have not yet been demonstrated. Objective To investigate the SCs of adult AL patients who were receiving chemotherapy and to determine their correlations with functional performance. Methods A total of 132 hospitalized adult AL patients were included in this study. A cross-sectional survey aimed to examine symptoms and functional performance was conducted. The patients' symptoms were assessed using the Chinese version of the Condensed Memorial Symptom Assessment Scale, and functional performance was evaluated through activities of daily living and quality of life. Results We identified 4 SCs in adult AL patients: psychological SC, pain-fatigue-sleep SC, dry mouth-constipation SC, and nutrition-impaired SC. The psychological SC was the most common and most distressing SC. The different SCs were each differentially correlated with patient characteristics. The distress of the psychological SC, pain-fatigue-sleep SC, and nutrition-impaired SC was adversely correlated with functional performance. Conclusions Adult AL patients undergoing chemotherapy experience multiple symptoms that can be further categorized into 4 SCs. The distress from some SCs is negatively associated with patients' functional performance. Implications for practice Symptom burden remains a major problem for adult AL patients undergoing chemotherapy. Identifying SCs of AL patients should be the basis for accurate and cost-effective interventions. Personalized SC management may improve the functional performance and healthcare quality of adult AL patients.

Osteolytic lesion (OL) and bone damage are common complications in multiple myeloma (MM). This study aimed to analyze the occurrence of OL in MM patient groups of different ages and genders.We performed a retrospective study of 762 MM patients admitted to West China Hospital from 2009 to 2014 to investigate the association between OL occurrence with patients' ages and genders. The presence or absence of OL was confirmed by X-ray, computed tomography (CT) or magnetic resonance imaging (MRI) examination. We also downloaded MM patients' published gene expression profiles and performed microarray-based analyses to identify differentially regulated genes and signaling pathways. Finally, we examined target gene expressions in MM bone marrow (BM) biopsies through immunohistochemistry (IHC).We calculated the frequency of OL in female and male MM patients with different age cut-offs. From West China Hospital data, we found that in young female MM patients aged under 55, the frequency of OL was 16.67%, significantly lower than the frequencies in other groups of patients (young males: 34.38%; old males: 31.04%; old females: 29.24%; p .05). The same was true in another independent MM cohort. Microarray-based analyses showed that Microtubule Associated Serine/Threonine Kinase Family Member 4 (MAST4), an estrogen-responsive gene, expression was up-regulated in MM patients without OL and in young female MM patients (p .05). The expression of MAST4 in MM BM was confirmed by IHC. The perspective of cell signaling network suggested that MAST4 might interact with phosphatase and tensin homolog (PTEN) and control the expression of a panel of osteoclast-regulatory cytokines, such as TNFSF11 and CCL2.Young female (55 years) MM patients have significantly lower OL frequency than other groups. MAST4 gene expression is thought to be associated with this phenomenon.

We retrospectively analyzed the samples collected from 66 patients with Ph+ALL enrolled on ChiCTR-TNRC-09000309 clinical trial. CR rate was 95.5%, and estimated 2-year OS and DFS were 51.7 ± 11.7% and 26.9 ± 11.6%, 3-year OS and DFS were 31.6 ± 12.0% and 23.4 ± 11.6%. By combining IKZF1 deletion and early molecular responses, we redefined the patients as low, intermediate, and high risk 3 groups separately. Patients with double negative in IKZF1 and early molecular response experienced significant superior survival, while patients with double positive would have the worst outcome, and patients who were one or the other with IKZF1 deletion or MRD status had intermediate outcome. Significant differences were found among 3 groups in regard to both OS (p

Our previous studies showed that macrophages (MФs), especially myeloma-associated MФs (MAMs), induce chemoresistance in human myeloma. Here we explored the potential of targeting MФs, by using colony-stimulating factor 1 receptor (CSF1R)-blocking mAbs, to treat myeloma. Our results showed that CSF1R blockade specifically inhibited the differentiation, proliferation and survival of murine M2 MФs and MAMs, and repolarized MAMs towards M1-like MФs in vitro. CSF1R blockade alone inhibited myeloma growth in vivo, by partially depleting MAMs, polarizing MAMs to the M1 phenotype, and inducing a tumor-specific cytotoxic CD4+ T-cell response. Similarly, genetically depleting MФs in myeloma-bearing MMDTR mice retarded myeloma growth in vivo. Furthermore, the combination of CSF1R blockade and chemotherapy such as bortezomib or melphalan displayed an additive therapeutic efficacy against established myeloma. Finally, a fully human CSF1R blocking mAb, similar to its murine counterpart, was able to inhibit the differentiation, proliferation and survival of human MФs. Thus, this study provides the first direct in vivo evidence that MΦs and MAMs are indeed important for myeloma development and progression. Our results also suggest that targeting MAMs by CSF1R blocking mAbs may be promising methods to (re)sensitize myeloma cells to chemotherapy and promote anti-myeloma immune responses in patients.

The aberrant expression or mutation of CFTR has been shown to be involved in several tumors, but how mutations or dysfunction of CFTR may increase the risk of malignancies in various tissues remains unclear. Here, we report the interaction between CFTR and HDAC2, and its involvement in the development of Ph+ leukemia. We first detected the physical interaction and co-localization of CFTR and HDAC2 in Ph+ leukemia cell lines. And treatment with CFTRinh-172, a CFTR inhibitor, reduced the expression of HDAC2 protein in K562 and SUP-B15 cells, which could be partially recovered by MG132, a proteasome inhibitor. Additionally, high expression levels of HDAC2 protein were observed in CFTR cDNA transfected HEK-293 and Ba/F3 cells. Next, we found that HDAC2 bound in the promoter region of the PTEN gene, and treatment with HDAC2 inhibitor or CFTRinh-172 resulted in an increase in PTEN mRNA and protein levels and a decrease in PDK1 and mTOR (downstream signaling of PTEN) activity. Finally, the MTT assay revealed that CFTRinh-172 could strengthen the anti-proliferation effect of HDAC2 inhibitor on Ph+ leukemia cells. Altogether, this study provides strong evidence that high-expression CFTR plays an important role in the development of Ph+ leukemia through the HDAC2-mediated PTEN pathway.

Background The physiological approach suggests that an environment associating mesenchymal stromal cells with low O2 concentration would be most favorable for the maintenance of hematopoietic stem/progenitor cells (HSPCs). To test this hypothesis, we performed a coculture of cord blood CD34+ cells with Wharton’s jelly mesenchymal stem cells (WJ-MSCs) under different O2 concentration to simulate the growth of HSPCs in vivo, and assessed the impacts on stemness maintenance and proliferation of cord blood HSPCs in vitro. Methods CD34+ cells derived from cord blood were isolated and cocultured under 1%, 3%, or 20% O2 concentrations with irradiated WJ-MSCs without adding exogenous cytokines for 7 days. The cultured cells were harvested and analyzed for phenotype and functionality, including total nuclear cells (TNC), CD34+Lin− cells, colony forming unit (CFU) for committed progenitors, and long-term culture initiating cells (LTC-ICs) for HSPCs. The cytokine levels in the medium were detected with Luminex liquid chips, and the mRNA expression of hypoxia inducible factor (HIF) genes and stem cell signal pathway (Notch, Hedgehog, and Wnt/β-catenin) downstream genes in cord blood HSPCs were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Results Our results showed that the number of TNC cells, CD34+Lin− cells, and CFU were higher or similar with 20% O2 (normoxia) in coculture and compared with 1% O2 (hypoxia). Interestingly, a 1% O2 concentration ensured better percentages of CD34+Lin− cells and LTC-IC cells. The hypoxia tension (1% O2) significantly increased vascular endothelial growth factor (VEGF) secretion and decreased interleukin (IL)-6, IL-7, stem cell factor (SCF), and thrombopoietin (TPO) secretion of WJ-MSCs, and selectively activated the Notch, Wnt/β-catenin, and Hedgehog signaling pathway of cord blood HSPCs by HIF-related factors, which may play an important role in stemness preservation and for sustaining HSPC quiescence. Conclusions Our data demonstrate that cord blood HSPCs maintain stemness better under hypoxia than normoxia with WJ-MSC coculture, partially due to the increased secretion of VEGF, decreased secretion of IL-6 by WJ-MSCs, and selective activation of stem cell signal pathways in HSPCs. This suggests that the oxygenation may not only be a physiological regulatory factor but also a cell engineering tool in HSPC research, and this may have important translational and clinical implications.

To study glycolysis/glycogenesis-related genes expression in childhood B-cell acute lymphoblastic leukaemia (B-ALL), we performed a microarray-based analysis using published gene expression profiles. We found that SLC2A5, which encodes solute carrier family 2 member 5 (SLC2A5, previously termed GLUT5) that facilitates cell fructose uptake, was up-regulated in Philadelphia chromosome-positive ALL (Ph+ALL). Microarray-based analyses also suggested that SLC2A5 expression was significantly down-regulated in childhood B-ALL with t(1;19) or 11q23 mutation. High SLC2A5 expression was found in patients who had disease recurrence within 3 years, early relapse, shortened complete remission duration and positive minimal residue disease (MRD) status after treatment. SLC2A5 overexpression at both the mRNA and protein level in Ph+ALL was confirmed in a validation cohort of childhood B-ALL. We also validated the correlation of SLC2A5 expression and MRD status. A mechanistic study using a human Ph+ALL cell line showed that BCR-ABL1 kinase might regulate SLC2A5 expression via MYC. The tyrosine kinase inhibitors (TKIs) imatinib and dasatinib repressed SLC2A5 expression and the cell uptake of fructose. Fructose protected the tumour cells from nutrition deficiency and drug-induced cell death. Overall, our findings showed that SLC2A5 was up-regulated in childhood Ph+ALL. SLC2A5 expression correlated with childhood B-ALL clinical factors, such as MRD status. Given that TKIs could inhibit SLC2A5 expression, repression of fructose utility after TKI treatment contributes to TKI-induced Ph+ALL cytotoxicity. Targeting SLC2A5 might be promising in B-ALL treatment, especially for Ph+ALL patients with high SLC2A5 expression.

PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the "verge of apoptosis". When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways.

// Yi Li 1,2,* , Yuhuan Zheng 2,3,* , Tianshu Li 2,* , Qiang Wang 2 , Jianfei Qian 2 , Yong Lu 2 , Mingjun Zhang 2 , Enguang Bi 2 , Maojie Yang 2 , Frederic Reu 4 , Qing Yi 2,** and Zhen Cai 1,** 1 Bone Marrow Transplantation Center, Department of Hematology, Zhejiang University, Hangzhou, Zhejiang, China 2 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 3 Department of Hematology, Sichuan University, West China School of Medicine, Chengdu, Sichuan, China 4 Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA * These authors have contributed equally to this manuscript ** These authors have contributed equally as senior authors to this manuscript Correspondence to: Zhen Cai, email: // Qing Yi, email: // Keywords : multiple myeloma, chemokine, macrophage, bone marrow Received : April 24, 2015 Accepted : June 05, 2015 Published : June 19, 2015 Abstract We previously showed that macrophages (MΦs) infiltrate the bone marrow (BM) of patients with myeloma and may play a role in drug resistance. This study analyzed chemokines expressed by myeloma BM that are responsible for recruiting monocytes to the tumor bed. We found that chemokines CCL3, CCL14, and CCL2 were highly expressed by myeloma and BM cells, and the levels of CCL14 and CCL3 in myeloma BM positively correlated with the percentage of BM-infiltrating MΦs. In vitro, these chemokines were responsible for chemoattracting human monocytes to tumor sites and in vivo for MΦ infiltration into myeloma-bearing BM in the 5TGM1 mouse model. Surprisingly, we also found that these chemokines stimulated MΦ in vitro proliferation induced by myeloma cells and in vivo in a human myeloma xenograft SCID mouse model. The chemokines also activated normal MΦ polarization and differentiation into myeloma-associated MΦs. Western blot analysis revealed that these chemokines promoted growth and survival signaling in MΦs via activating the PI3K/Akt and ERK MAPK pathways and c-myc expression. Thus, this study provides novel insight into the mechanism of MΦ infiltration of BM and also potential targets for improving the efficacy of chemotherapy in myeloma.

// Mingjun Zhang 1, 2 , Jin He 1 , Zhiqiang Liu 1 , Yong Lu 1, 2 , Yuhuan Zheng 1, 2 , Haiyan Li 1, 2 , Jingda Xu 1 , Huan Liu 1 , Jianfei Qian 1, 2 , Robert Z. Orlowski 1 , Larry W. Kwak 1 , Qing Yi 1, 2 , Jing Yang 1, 3 1 Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA 3 Cancer Research Institute and Cancer Hospital, Guangzhou Medical University, Guangzhou, China Correspondence to: Jing Yang, e-mail: jiyang@mdanderson.org Qing Yi, e-mail: yiq@ccf.org Keywords: multiple myeloma, anti-β 2 M monoclonal antibody, bortezomib, autophagy, NF-κ p65 Received: November 17, 2014 Accepted: January 29, 2015 Published: March 31, 2015 ABSTRACT Our previous studies showed that anti-β 2 M monoclonal antibodies (mAbs) have strong and direct apoptotic effects on multiple myeloma (MM) cells, suggesting that anti-β 2 M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the anti-MM effects of combination treatment with anti-β 2 M mAbs and bortezomib (BTZ). Our results showed that anti-β 2 M mAbs enhanced BTZ-induced apoptosis of MM cell lines and primary MM cells. Combination treatment could also induce apoptosis of BTZ-resistant MM cells, and the enhanced effect depended on the surface expression of β 2 M on MM cells. BTZ up-regulated the expression of autophagy proteins, whereas combination with anti-β 2 M mAbs inhibited autophagy. Sequence analysis of the promoter region of beclin 1 identified 3 putative NF-κB-binding sites from –615 to –789 bp. BTZ treatment increased, whereas combination with anti-β 2 M mAbs reduced, NF-κB transcription activities in MM cells, and combination treatment inhibited NF-κB p65 binding to the beclin 1 promoter. Furthermore, anti-β 2 M mAbs and BTZ combination treatment had anti-MM activities in an established MM mouse model. Thus, our studies provide new insight and support for the clinical development of an anti-β 2 M mAb and BTZ combination treatment to overcome BTZ drug resistance and improve MM patient survival.

// Xi Yang 1, * , Tianyou Yan 1, * , Yuping Gong 1 , Xuehua Liu 1 , Huaqin Sun 2 , Wenming Xu 2 , Chunsen Wang 3 , Duolan Naren 1 , Yuhuan Zheng 1 1 Department of Hematology, West China Hospital, Sichuan University, Chengdu, China 2 Sichuan University-The Chinese University of Hong Kong Joint Laboratory for Reproductive Medicine, West China Second University Hospital, Chengdu, China 3 Department of Hematology, Sichuan Provincial People’s Hospital, Affiliated Hospital of University of Electronic Science and Technology of China, Chengdu, China * These authors contributed equally to this work Correspondence to: Yuping Gong, email: gongyuping2010@aliyun.com Keywords: CFTR, Ph + acute leukemia, PP2A Received: May 30, 2016 Accepted: February 06, 2017 Published: February 19, 2017 ABSTRACT Cystic fibrosis transmembrane conductance regulator (CFTR) is classified as an anion channel transporter of Cl − and HCO3 − . Through interactions with its PDZ domain, CFTR is capable of regulating other proteins, such as protein phosphatase 2A (PP2A). The aberrant expression and mutation of CFTR have been observed in several tumor, but not in philadelphia chromosome–positive(Ph + ) acute leukemia, including Ph + B cell acute lymphoblastic leukemia(Ph + B-ALL) and chronic myelogenous leukemia blast crisis phases (CML-BC). In this study, we demonstrated the mean expression level of CFTR in Ph + acute leukemia cells was markedly higher than that in Ph- B-ALL and CML-chronic phase cells. CFTRinh-172, a classic CFTR inhibitor, down-regulated the expression of CFTR, p-BCR-ABL and classical Wnt/β-catenin signaling in Ph + acute leukemia cells, while imatinib had no effect on CFTR. Importantly, reduced efficacy of CFTRinh-172 was closely associated with elevated PP2A phosphatase activity. Furthermore, we confirmed an interaction between CFTR and the PP2AA subunit in K562 cells. In addition, we demonstrated CFTR and PP2AA interact in the cytosol, resulting in PP2 A complex inactivation and increased degradation of PP2 A substrates via the lysosomal/proteasome pathway. In conclusion, our results showed CFTR was highly expressed in Ph + acute leukemia, which protected and maintained the continuous activation of BCR-ABL and the canonical Wnt/β-catenin signaling pathway by decreasing PP2A phosphatase activity. According to this working model of the CFTR-PP2A-BCR-ABL axis, targeting the CFTR protein will activate PP2A and may offer a new treatment strategy for Ph + acute leukemia, especially for patients exhibiting high levels of CFTR expression.

Mammalian target of rapamycin (mTOR) signaling is a central regulator of protein translation, cell growth, and metabolism. Alterations of the mTOR signaling pathway are common in cancer, making mTOR a promising therapeutic target. In clinical trials, rapamycin analogs have shown modest response rates for most cancer types, including breast cancer. Therefore, there is an urgent need to better understand the mechanism of action of rapamycin to improve patient selection and to monitor pathway inhibition. To identify novel pharmacodynamic markers of rapamycin activity, we carried out transcriptional profiling of total and polysome-associated RNA in three breast cancer cell lines representing different subtypes. In all three cell lines, we found that rapamycin significantly decreased polysome-associated mRNA for stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis. Activators of mTOR increased SCD1 protein expression, whereas rapamycin, LY294002, and BEZ235 decreased SCD1 protein expression. Rapamycin decreased total SCD1 RNA expression without inducing a significant decline in its relative polysomal recruitment (polysome/total ratio). Rapamycin did not alter SCD1 mRNA stability. Instead, rapamycin inhibited SCD1 promoter activity and decreased expression of mature transcription factor sterol regulatory element binding protein 1 (SREBP1). Eukaryotic initiation factor 4E (eIF4E) small interfering RNA (siRNA) decreased both SCD1 and SREBP1 expression, suggesting that SCD1 may be regulated through the mTOR/eIF4E-binding protein 1 axis. Furthermore, SCD1 siRNA knockdown inhibited breast cancer cell growth, whereas overexpression increased growth. Taken together these findings show that rapamycin decreases SCD1 expression, establishing an important link between cell signaling and cancer cell fatty acid synthesis and growth. Mol Cancer Ther; 9(10); 2770–84. ©2010 AACR.

The bone and immune systems are closely related through cellular and molecular interactions. Because bone-resorbing osteoclasts (OCs) are derived from the monocyte/macrophage lineage, similar to dendritic cells (DCs), we hypothesized that OCs could serve as antigen-presenting cells (APCs) to activate T cells. In this study, OCs were generated from human monocytes with stimulation by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Results showed that, similar to DCs, OCs express major histocompatibility complex (MHC) classes I and II, and CD80, CD86, and CD40; and uptake soluble antigens. OCs secrete interleukin-10 (IL-10), transforming growth factor-β (TGF-β), IL-6, and tumor necrosis factor-α (TNF-α), but not IL-12p70. OCs present allogeneic antigens and activate both CD4+ and CD8+ alloreactive T cells in an MHC-restricted fashion. OCs also present soluble protein tetanus toxoid to activate autologous CD4+ T cells. These findings indicate that OCs can function as APCs and activate both CD4+ and CD8+ T cells. Thus, our study provides new insight into the effect of OCs on the immune system and may help develop novel strategies for treating diseases such as rheumatoid arthritis and multiple myeloma, which affect both the bone and immune systems.

Multiple myeloma remains an incurable disease. One of the major problems is that myeloma cells develop drug resistance on interaction with bone marrow stromal cells. In this study, we examined the effects of macrophages (Mφs), a type of stromal cells, on myeloma cell survival and response to chemotherapy. We showed that Mφ, in particular tumor-associated Mφ, is a protector of myeloma cells. The protective effect was dependent on direct contact between Mφs and myeloma cells. Mφs protected both myeloma cell lines and primary myeloma cells from spontaneous and chemotherapy drug-induced apoptosis by attenuating the activation and cleavage of caspase-dependent apoptotic signaling. These findings are clinically relevant because we found that CD68+ Mφs heavily infiltrate the bone marrow of patients with myeloma but not the bone marrow of control patients. Thus, our results indicate that Mφs may contribute to myeloma cell survival and resistance to chemotherapeutic treatment in vivo.

Hedgehog (Hh) signaling plays an important role in the oncogenesis of B-cell malignancies such as multiple myeloma (MM). However, the source of Hh ligand sonic hedgehog (SHH) and its target cells remains controversial. Previous studies showed that stromally induced Hh signaling is essential for the tumor cells and that CD19(+)CD138(-) MM stem cells are the target cells of Hh signaling. Here we demonstrate that SHH was mainly secreted by human myeloma cells but not by stromal cells in MM bone marrow. Autocrine SHH enhanced CD138(+) myeloma cell proliferation and protected myeloma cells from spontaneous and stress-induced apoptosis. More importantly, autocrine SHH protected myeloma cells against chemotherapy-induced apoptosis in vitro and in vivo. Combinational treatment with chemotherapy and SHH-neutralizing antibody displayed synergistic antimyeloma effects. Mechanistic studies showed that SHH signaling activated the SHH/GLI1/BCL-2 axis, leading to the inhibition of myeloma cell apoptosis. Thus, this study identifies the myeloma autocrine Hh signaling pathway as a potential target for the treatment of MM. Targeting this pathway may improve the efficacy of chemotherapy in MM patients.

Dendritic cells (DCs) are professional antigen-presenting cells to initiate immune responses, and DC survival time is important for affecting the strength of T-cell responses. Interleukin (IL)-9-producing T-helper (Th)-9 cells play an important role in anti-tumor immunity. However, it is unclear how Th9 cells communicate with DCs. In this study, we investigated whether murine Th9 cells affected the survival of myeloid DCs. DCs derived from bone marrow of C57BL/6 mice were cocultured with Th9 cells from OT-II mice using transwell, and the survival of DCs was examined. DCs cocultured with Th9 cells had longer survival and fewer apoptotic cells than DCs cultured alone in vitro. In melanoma B16-OVA tumor-bearing mice, DCs conditioned by Th9 cells lived longer and induced stronger anti-tumor response than control DCs did in vivo. Mechanistic studies revealed that IL-3 but not IL-9 secreted by Th9 cells was responsible for the prolonged survival of DCs. IL-3 upregulated the expression of anti-apoptotic protein Bcl-xL and activated p38, ERK and STAT5 signaling pathways in DCs. Taken together, our data provide the first evidence that Th9 cells can promote the survival of DCs through IL-3, and will be helpful for designing Th9 cell immunotherapy and more effective DC vaccine for human cancers.

Introduction and objectives: Cervical cancer is the only gynaecological cancer that is staged clinically. The clinical stage of cervical cancer relies largely on the pelvic examination. The aim of this study is to analyse the discrepancy between clinical stage and pathological results, and to explore the accuracy of pelvic examination. Methods: We collected retrospective data from 818 patients with cervical carcinoma staged IB–IIB, who were treated with primary surgery from January 1999 to June 2007. Clinical stages of those patients were determined by pelvic examination without anaesthesia. After surgery, all the patients were assigned to pT category according to the pathological findings. Comparisons were made between these two stages. Results: The total concordance between clinical stage and pT category for stage IB–IIB was 53.1%, with an overestimation of 37.3% and an underestimation of 9.7%. The concordance in stage IB1, stage IB2, stage IIA and stage IIB were 85.4%, 77.4%, 35.3% and 20.5%, respectively. The most significant discrepancy was caused by the failure to detect the parametrial invasion accurately in stage IIB. The accuracy of pelvic examination to determine vaginal and parametrial disease was 70.2% and 74.0%, respectively. Conclusions: There are significant discrepancies between clinical stage and pathological results. Pelvic examination has its limitations in staging determination. Thus for operable cervical cancer, clinical stage alone is not reliable for selecting postoperative therapies and surgical staging system may be considered.

Osteoclasts (OCs) are bone resorbing cells whose activity can be regulated by activated T cells and their cytokines. However, the immune function of OCs is largely unknown. In this study, we found that as bystanders, human OCs effectively suppressed T-cell proliferation induced by allogeneic, microbial antigenic and T-cell receptor stimuli in vitro. Mechanistic studies revealed that T cell-derived IFN-γ and CD40 ligand (CD40L) induced the expression of indoleamine 2,3-dioxygenase (IDO) in OCs, which mediated the immunosuppressive function on T-cell proliferation through depleting tryptophan. Neutralizing IFN-γ and blocking CD40L, and silencing or inhibiting IDO in OCs restored T-cell proliferation in the presence of OCs. Our data reveal a novel function of human OCs as inducible immunosuppressive cells, and a feedback loop between OCs and activated T cells. Thus, this study provides new insight into the mechanism of the immunosuppressive function of OCs, and may be helpful for developing novel therapeutic strategies for human diseases involving both the bone and immune systems.

Dendritic cell (DC)-based cancer immunotherapy is a promising method, but so far has demonstrated limited clinical benefits. Regulatory T cells (Tregs) represent a major obstacle to cancer immunotherapy approaches. Here we show that inhibiting p38 MAPK during DC differentiation enables DCs to activate tumour-specific effector T cells (Teff), inhibiting the conversion of Treg and compromising Treg inhibitory effects on Teff. Inhibition of p38 MAPK in DCs lowers expression of PPARγ, activating p50 and upregulating OX40L expression in DCs. OX40L/OX40 interactions between DCs and Teff and/or Treg are critical for priming effective and therapeutic antitumour responses. Similarly, p38 MAPK inhibition also augments the T-cell stimulatory capacity of human monocyte-derived DCs in the presence of Treg. These findings contribute to ongoing efforts to improve DC-based immunotherapy in human cancers.

Our previous studies showed that anti-β2M monoclonal antibodies (mAbs) at high doses have direct apoptotic effects on myeloma cells, suggesting that anti-β2M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the ability of the mAbs at much lower concentrations to indirectly kill myeloma cells by utilizing immune effector cells or molecules. Our results showed that anti-β2M mAbs effectively lysed MM cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which were correlated with and dependent on the surface expression of β2M on MM cells. The presence of MM bone marrow stromal cells or addition of IL-6 did not attenuate anti-β2M mAb-induced ADCC and CDC activities against MM cells. Furthermore, anti-β2M mAbs only showed limited cytotoxicity toward normal B cells and non-tumorous mesenchymal stem cells, indicating that the ADCC and CDC activities of the anti-β2M mAbs were more prone to the tumor cells. Lenalidomide potentiated in vitro ADCC activity against MM cells and in vivo tumor inhibition capacity induced by the anti-β2M mAbs by enhancing the activity of NK cells. These results support clinical development of anti-β2M mAbs, both as a monotherapy and in combination with lenalidomide, to improve MM patient outcome.

Clinical trials of immunotherapy in mantle cell lymphoma have not yet delivered desirable results, partly because of the inhibitory machinery of the tumor and its microenvironment. Here we investigated the role of B7-H1, a member of the B7 family of co-stimulatory/co-inhibitory ligands, in mantle cell lymphoma-mediated immunosuppression. Allogeneic CD3(+), CD4(+) and CD8(+) T cells were purified and co-cultured with irradiated mantle cell lymphoma cells. Mantle cell lymphoma-reactive T-cell lines from HLA-A*0201(+) healthy blood donors were generated after in vitro restimulation, and were subjected to functional tests. We found that B7-H1 expressed on mantle cell lymphoma cells was able to inhibit T-cell proliferation induced by the tumor cells, impair the generation of antigen-specific T-cell responses, and render mantle cell lymphoma cells resistant to T-cell-mediated cytolysis. Blocking or knocking down B7-H1 on mantle cell lymphoma cells enhanced T-cell responses and restored tumor-cell sensitivity to T-cell-mediated killing in vitro and in vivo. Knocking down B7-H1 on mantle cell lymphoma cells primed more CD4(+) or CD8(+) memory effector T cells. Our study demonstrates for the first time that lymphoma cell-expressed B7-H1 may lead to the suppression of host anti-tumor immune responses in mantle cell lymphoma and targeting tumor cell B7-H1 may represent a novel approach to improve the efficacy of immunotherapy in patients with mantle cell lymphoma.

BACKGROUND Multiple myeloma (MM) remains an incurable cancer characterized by accumulation of malignant plasma cells in the bone marrow (BM). The mechanism underlying MM homing to BM is poorly elucidated. METHODS The clinical significance of migration inhibitory factor (MIF) expression was examined by analyzing six independent gene expression profile databases of primary MM cells using the Student's t test and Kaplan-Meier test. Enzyme-linked immunosorbent assay was used to examine MIF expression. In vivo bioluminescent imaging was used to determine MM cell localization and treatment efficacy in human MM xenograft mouse models, with three to four mice per group. MM cell attachment to BM stromal cells (BMSCs) was monitored by cell adhesion assay. MIF regulation of the expression of adhesion molecules was determined by chromatin immunoprecipitation (ChIP) assay. Statistical tests were two-sided. RESULTS High levels of MIF were detected in MM BM (MIF level in BM plasma: healthy = 10.72 ± 5.788 ng/mL, n = 5; MM = 1811 ± 248.7 ng/mL, n = 10; P < .001) and associated with poor survival of patients (Kaplan-Meier test for MM OS: 87 MIF(high) patients, 86 MIF(low) patients, P = .02). Knocking down MIF impaired MM cell adhesion to BMSCs in vitro and led to formation of extramedullary tumors in SCID mice. MIF acted through surface receptor CXCR4 and adaptor COPS5 to regulate the expression of adhesion molecules ALCAM, ITGAV, and ITGB5 on MM cells. More importantly, MIF-deficient MM cells were sensitive to chemotherapy in vitro when cocultured with BMSCs and in vivo. MIF inhibitor 4-IPP sensitized MM cells to chemotherapy. CONCLUSIONS MIF is an important player and a novel therapeutic target in MM. Inhibiting MIF activity will sensitize MM cells to chemotherapy.

p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in human myeloma, has been implicated in bone destruction by this cancer, but the processes it recruits are obscure. In this study, we show that p38 activity in myeloma inhibits osteoblast differentiation and bone formation, but also enhances osteoclast maturation and bone resorption. p38 regulated the expression and secretion of the Wnt pathway antagonist DKK-1 and the monocyte chemoattractant MCP-1. Attenuating p38, DKK-1, or MCP-1 were each sufficient to reduce bone lesions in vivo. Although it is well known that DKK-1 inhibits osteoblast differentiation, we found that together with MCP-1, it could also promote osteoclast differentiation and bone resorption. The latter effects were mediated by enhancing expression of RANK in osteoclast progenitor cells and by upregulating secretion of its ligand RANKL from stromal cells and mature osteoblasts. In summary, our study defined the mechanisms by which p38 signaling in myeloma cells regulates osteoblastogenesis, osteoclastogenesis, and bone destruction. Our findings, which may have implications for bone invasion by other cancers where p38 is elevated, strongly suggests that targeting p38 for inhibition may offer an effective therapeutic approach to treat osteolytic bone lesions in patients with myeloma. Cancer Res; 72(24); 6393–402. ©2012 AACR.

Chemoresistance is the major obstacle in multiple myeloma (MM) management. We previously showed that macrophages protect myeloma cells, on a cell contact basis, from melphalan or dexamethasone-induced apoptosis in vitro. In this study, we found that macrophage-mediated myeloma drug resistance was also seen with purified macrophages from myeloma patients’ bone marrow (BM) in vitro and was confirmed in vivo using the human myeloma-SCID (severe combined immunodeficient) mouse model. By profiling differentially regulated and paired plasma membrane protein genes, we showed that PSGL-1 (P-selectin glycoprotein ligand-1)/selectins and ICAM-1/CD18 played an important role in macrophage-mediated myeloma cell drug resistance, as blocking antibodies against these molecules or genetic knockdown of PSGL-1 or ICAM-1 in myeloma cells repressed macrophages’ ability to protect myeloma cells. Interaction of macrophages and myeloma cells via these molecules activated Src and Erk1/2 kinases and c-myc pathways and suppressed caspase activation induced by chemotherapy drugs. Thus, our study sheds new light on the mechanism of drug resistance in MM and provides novel targets for improving the efficacy of chemotherapy in patients.

Idiotype (Id) protein in combination with GM-CSF has been used as vaccines for immunotherapy of patients with myeloma and B-cell tumors and the results have been disappointing. To search for better immune adjuvants to improve the efficacy of Id-based immunotherapy in myeloma, we evaluated and compared the efficacy of vaccination of Id protein in combination with CpG or IFN-α, or GM-CSF as a control, in the 5TGM1 myeloma mouse model. Our results showed that Id vaccine combined with CpG or IFN-α, but not GM-CSF, not only efficiently protected mice from developing myeloma but also eradicated established myeloma. The therapeutic responses were associated with an induction of strong humoral immune responses including anti-Id antibodies, and cellular immune responses including Id- and myeloma-specific CD8+ cytotoxic T lymphocytes (CTLs), CD4+ type-1 T-helper (Th1) cells and memory T cells in mice receiving Id vaccine combined with CpG or IFN-α. Furthermore, Id vaccine combined with CpG or IFN-α induced Id- and tumor-specific memory immune responses that protected surviving mice from tumor rechallenge. Thus, our study clearly shows that CpG or IFN-α are better immune adjuvants than GM-CSF. This information will be important for improving the strategies of Id-based immunotherapy for patients with myeloma and other B-cell tumors.

NVP-BKM120 is a novel phosphatidylinositol 3-kinase (PI3K) inhibitor and is currently being investigated in phase I clinical trials in solid tumors. This study aimed to evaluate the therapeutic efficacy of BKM120 in multiple myeloma (MM). BKM120 induces cell growth inhibition and apoptosis in both MM cell lines and freshly isolated primary MM cells. However, BKM120 only shows limited cytotoxicity toward normal lymphocytes. The presence of MM bone marrow stromal cells, insulin-like growth factor, or interleukin-6 does not affect BKM120-induced tumor cell apoptosis. More importantly, BKM120 treatment significantly inhibits tumor growth in vivo and prolongs the survival of myeloma-bearing mice. In addition, BKM120 shows synergistic cytotoxicity with dexamethasone in dexamethasone-sensitive MM cells. Low doses of BKM120 and dexamethasone, each of which alone has limited cytotoxicity, induce significant cell apoptosis in MM.1S and ARP-1. Mechanistic study shows that BKM120 exposure causes cell cycle arrest by upregulating p27 (Kip1) and downregulating cyclin D1 and induces caspase-dependent apoptosis by downregulating antiapoptotic XIAP and upregulating expression of cytotoxic small isoform of Bim, BimS. In summary, our findings demonstrate the in vitro and in vivo anti-MM activity of BKM120 and suggest that BKM120 alone or together with other MM chemotherapeutics, particularly dexamethasone, may be a promising treatment for MM.

Monoclonal antibodies (mAbs) specific for human β(2) -microglobulin (β(2) M) have been shown to induce tumour cell apoptosis in haematological and solid tumours via recruiting major histocompatibility complex (MHC) class I molecules into and excluding cytokine receptors from the lipid rafts. Based on these findings, we hypothesized that IgM anti-β(2) M mAbs might have stronger apoptotic effects because of their pentameric structure. Our results showed that, compared with IgG mAbs, IgM anti-β(2) M mAbs exhibited stronger tumouricidal activity in vitro against different tumour cells, including myeloma, mantle cell lymphoma, and prostate cancer, and in vivo in a human-like xenografted myeloma mouse model without damaging normal tissues. IgM mAb-induced apoptosis is dependent on the pentameric structure of the mAbs. Disrupting pentameric IgM into monomeric IgM significantly reduced their ability to induce cell apoptosis. Monomeric IgM mAbs were less efficient at recruiting MHC class I molecules into and exclusion of cytokine receptors from lipid rafts, and at activating the intrinsic apoptosis cascade. Thus, we developed and validated the efficacy of anti-β(2) M IgM mAbs that may be utilized in the clinical setting and showed that IgM anti-β(2) M mAbs may be more potent than IgG mAbs at inducing tumour apoptosis.

The cyclin dependent kinase inhibitor p27 (Kip1) plays an important role in controlling the eukaryotic cell cycle. The 5'-untranslated region of the p27 mRNA harbors an internal ribosome entry site (IRES) which may facilitate synthesis of p27 in certain conditions. In this study, the RNA-associated protein CUGBP1 was shown to interact with the human p27 5'-untranslated region. Overexpression of CUGBP1 inhibited endogenous p27 expression and reduced translation initiation through the p27 IRES. In contrast, repression of CUGBP1 by siRNA transfection enhanced p27 protein levels and stimulated p27 IRES activity. Addition of recombinant CUGBP1 repressed p27 IRES reporter mRNA translation in vitro. At last, Our finding showed that cytosolic form of CUGBP1 binds efficiently to the p27 5'-untranslated region.

The cyclin dependent kinase inhibitor p27 plays an important role in controlling the eukaryotic cell cycle by regulating progression through G1 and entry into S phase. It is often elevated during differentiation and under conditions of cellular stress. In contrast, it is commonly downregulated in cancer cells and its levels are generally inversely correlated with favorable prognosis. The cellular levels of p27 are regulated, in part, by translational control mechanisms. The 5′-untranslated region (5′-UTR) of the p27 mRNA harbors an internal ribosome entry site (IRES) which may facilitate synthesis of p27 in certain conditions. In this study, Far Upstream Element (FUSE) Binding Protein 1 (FBP1) was shown to directly bind to the human p27 5′-UTR and to promote IRES activity. An eight-nucleotide element downstream of a U-rich region within the 5′-UTR was important for FBP1 binding and p27 IRES activity. Overexpression of FBP1 enhanced endogenous p27 levels and stimulated translation initiation. In contrast, repression of FBP1 by siRNA transfection downregulated endogenous p27 protein levels. Using rabbit reticulocyte lysates, FBP1 stimulated p27 mRNA translation in vitro. The central domain of FBP1, containing four K homology motifs, was required for p27 5′-UTR RNA binding and the N terminal domain was important for translational activation. These findings indicate that FBP1 is a novel activator of p27 translation upon binding to the 5′-UTR.

Activation of translation initiation is essential for the malignant phenotype and is emerging as a potential therapeutic target. Translation is regulated by the expression of translation initiation factor 4E (eIF4E) as well as the interaction of eIF4E with eIF4E-binding proteins (e.g., 4E-BP1). Rapamycin inhibits translation initiation by decreasing the phosphorylation of 4E-BP1, increasing eIF4E/4E-BP1 interaction. However, rapamycin also inhibits S6K phosphorylation, leading to feedback loop activation of Akt. We hypothesized that targeting eIF4E directly would inhibit breast cancer cell growth without activating Akt. We showed that eIF4E is ubiquitously expressed in breast cancer cell lines. eIF4E knockdown by small interfering RNA inhibited growth in different breast cancer cell subtypes including triple-negative (estrogen receptor/progesterone receptor/HER-2–negative) cancer cells. eIF4E knockdown inhibited the growth of cells with varying total and phosphorylated 4E-BP1 levels and inhibited rapamycin-insensitive as well as rapamycin-sensitive cell lines. eIF4E knockdown led to a decrease in expression of cyclin D1, Bcl-2, and Bcl-xL. eIF4E knockdown did not lead to Akt phosphorylation but did decrease 4E-BP1 expression. We conclude that eIF4E is a promising target for breast cancer therapy. eIF4E-targeted therapy may be efficacious in a variety of breast cancer subtypes including triple-negative tumors for which currently there are no targeted therapies. Unlike rapamycin and its analogues, eIF4E knockdown is not associated with Akt activation. [Mol Cancer Ther 2008;7(7):1782–8]

Multiple myeloma (MM) cells are responsible for aberrant osteoclast (OC) activation. However, when cocultured monocytes, but not OC precursors, with MM cells, we made a novel observation that MM cells inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced increase of OC differentiation, OC gene expression, signaling pathways and bone resorption activity. Our results showed that MM cells produced multiple inhibitory cytokines of osteoclastogenesis, such as IL-10, which activated STAT3 signaling and induce OC inhibition. However, cocultures of bone marrow stromal cells (BMSCs) reversed MM-induced OC inhibition. We found that MM cells increased production of MCP-1 from BMSCs and BMSC-derived MCP-1 enhanced OC formation. Mechanistic studies showed that IL-10 downregulated RANK expression in monocytes and thus, inhibited RANKL-induced OC formation. In contrast, MCP-1 upregulated RANK expression and thus, enhanced OC formation. Overall, our studies for the first time demonstrated that MM cell have inhibitory effects on osteoclastogenesis by producing inhibitory cytokines. Our results further indicate that activation of osteoclastogenesis in bone marrow requests the crosstalk of MM cells, BMSCs and their produced cytokines. Thus, our studies provide evidences that targeting bone marrow microenvironmental cells and/or cytokines may be a new approach to treating MM bone destruction.

Background Previous research suggested that single gene expression might be correlated with acute myeloid leukemia (AML) survival. Therefore, we conducted a systematical analysis for AML prognostic gene expressions. Methods We performed a microarray-based analysis for correlations between gene expression and adult AML overall survival (OS) using datasets GSE12417 and GSE8970. Positive findings were validated in an independent cohort of 50 newly diagnosed, non-acute promyelocytic leukemia (APL) AML patients by quantitative RT-PCR and survival analysis. Results Microarray-based analysis suggested that expression of eight genes was each associated with 1-year and 3-year AML OS in both GSE12417 and GSE8970 datasets (p