Side Population Research Articles

Temozolomide abrogates the aggressiveness of urothelial carcinoma cells by enhancing senescence and depleting the side population.

Patients with advanced urothelial carcinoma (UC) generally have poor prognoses due to therapeutic resistance. Furthermore, there are limited treatment options for advanced UC. Therefore, novel or effective chemotherapeutic agents are needed to improve patient survival. The present study was conducted to investigate the effect of temozolomide (TMZ) on UC cells so as to identify a potential method to overcome therapeutic resistance. TMZ is an alkylating agent with a target different from that of other anticancer drugs used to treat UC, such as cisplatin. TMZ enhanced the autophagic response and senescence, which was mediated via the p53 and p21 pathways. Inhibiting the autophagic response using chloroquine synergistically augmented the cytotoxic effect of TMZ on UC cells. TMZ significantly reduced the invasiveness of UC cells. Notably, the abundance of side population fraction was also significantly reduced following TMZ treatment. Considering that side population fraction is known to confer therapeutic resistance, it is noteworthy that the TMZ treatment markedly decreased side population fraction. Altogether, TMZ may have the potential to be applied as a part of an alternative treatment strategy to reduce the malignancy of UC cells.

Gastric cancer stem cells survive in stress environments via their autophagy system

Cancer stem cells (CSCs) play an important role in the progression of carcinoma and have a high potential for survival in stress environments. However, the mechanisms of survival potential of CSCs have been unclear. The aim of this study was to clarify the significance of autophagy systems of CSCs under stress environments. Four gastric cancer cell line were used. Side population (SP) cells were sorted from the parent cells, as CSC rich cells. The expression of stem cell markers was examined by RT-PCR. The viability of cancer cells under starvation and hypoxia was evaluated. The expression level of the autophagy molecule LC3B-II was examined by western blot. The numbers of autophagosomes and autolysosomes were counted by electron microscope. SP cells of OCUM-12 showed a higher expression of stem cell markers and higher viability in starvation and hypoxia. Western blot and electron microscope examinations indicated that the autophagy was more induced in SP cells than in parent cells. The autophagy inhibitor significantly decreased the viability under the stress environments. These findings suggested that Cancer stem cells of gastric cancer might maintain their viability via the autophagy system. Autophagy inhibitors might be a promising therapeutic agent for gastric cancer.

Bystander effect of ultraviolet A radiation protects A375 melanoma cells by induction of antioxidant defense

Ultraviolet (UV) irradiated cells release factors that result in varied responses by non-irradiated cells via bystander effects (BE). The UV-BE is dependent on the cell types involved and on the wavelength of the radiation. Using conditioned medium from UVA-irradiated A375 human melanoma cells (UVA-CM), UVA-bystander response was evaluated on the viability of naïve A375 cells. UVA-CM treatment itself did not alter cell viability; however, UVA-CM treated bystander cells were more resistant to the lethal action of UVA, UVB, UVC or H2O2. Effects of UVA-CM on cell proliferation, mechanism of cell death, DNA damage, malondialdehyde formation, generation of reactive oxygen species (ROS) and antioxidant status were studied in A375 cells. We observed that UVA-CM triggered antioxidant defenses to elicit protective responses through elevation of antioxidant enzyme activities in cells, which persisted until 5 h after exposure to UVA-CM. This was possibly responsible for decreased generation of ROS and diminished DNA and membrane damage in cells. These bystander cells were resistant to killing when exposed to different genotoxic agents. Damaged nuclei, induction of apoptosis and autophagic death were also lowered in these cells. The influence of UVA-CM on cancer stem cells side population was assessed. Highlights: UVA radiation induced bystander effects in A375 cells Damage by genotoxicants is suppressed due to lower ROS generation on UVA-CM treatment UVA-CM exposure enhanced higher activities of CAT and GPx Resistance to genotoxic agents in such cells was due to elevated antioxidant defence UVA-bystander phenomenon was a protective response

Combinatorial Effect of PLK1 Inhibition with Temozolomide and Radiation in Glioblastoma.

Simple SummaryThere is a critical need to identify readily translatable adjuncts to potentiate the dismal median survivals of only 15–20 months in glioblastoma (GBM) patients after standard of care, i.e., concurrent Temozolomide (TMZ) and radiation (XRT) therapy. Here we demonstrated that the Polo-like kinase 1 (PLK1) inhibitor volasertib, which has been employed in cancer clinical trials, has activity against GBM in the contexts of both as monotherapy and as an adjunct to standard of care (SOC). In addition to corroborating the known effects of volasertib, we found novel impacts of volasertib on mitochondrial membrane potential, ROS generation, persistent DNA damage and signaling pathways such as ERK/MAPK, AMPK and glucocorticoid receptor. Together these studies support the potential importance of PLK1 inhibitors as an adjunct to GBM SOC therapy that warrants further preclinical investigation.New strategies that improve median survivals of only ~15–20 months for glioblastoma (GBM) with the current standard of care (SOC) which is concurrent temozolomide (TMZ) and radiation (XRT) treatment are urgently needed. Inhibition of polo-like kinase 1 (PLK1), a multifunctional cell cycle regulator, overexpressed in GBM has shown therapeutic promise but has never been tested in the context of SOC. Therefore, we examined the mechanistic and therapeutic impact of PLK1 specific inhibitor (volasertib) alone and in combination with TMZ and/or XRT on GBM cells. We quantified the effects of volasertib alone and in combination with TMZ and/or XRT on GBM cell cytotoxicity/apoptosis, mitochondrial membrane potential (MtMP), reactive oxygen species (ROS), cell cycle, stemness, DNA damage, DNA repair genes, cellular signaling and in-vivo tumor growth. Volasertib alone and in combination with TMZ and/or XRT promoted apoptotic cell death, altered MtMP, increased ROS and G2/M cell cycle arrest. Combined volasertib and TMZ treatment reduced side population (SP) indicating activity against GBM stem-like cells. Volasertib combinatorial treatment also significantly increased DNA damage and reduced cell survival by inhibition of DNA repair gene expression and modulation of ERK/MAPK, AMPK and glucocorticoid receptor signaling. Finally, as observed in-vitro, combined volasertib and TMZ treatment resulted in synergistic inhibition of tumor growth in-vivo. Together these results identify new mechanisms of action for volasertib that provide a strong rationale for further investigation of PLK1 inhibition as an adjunct to current GBM SOC therapy.

Current status, challenges and perspectives of mesenchymal stem cell-based therapy for cardiac regeneration

Modern methods of treating heart failure are similar to the palliative care, since they mostly relieve the symptoms of the disease. The discovery of resident cardiac stem cells gave impetus to the development of “second generation” cell therapy, which quickly moved from animal research to clinical trials with critically ill patients. Many cardiac side population cells have been identified to have stem cells characteristics and some additional individual characteristics, both in vitro and in vivo. The results of clinical studies demonstrated that the stem cell treatment is safe, however, this type of cell-based therapy did not restore cardiac function. Its effects were limited to mildly improving left ventricular systolic pressure and reducing the scar area. Despite that, the promising nature of these therapeutic approaches for heart diseases have contributed to the development of next-generation cell therapy.

A direct negative feedback loop of miR-4721/FOXA1/Nanog promotes nasopharyngeal cell stem cell enrichment and metastasis

ObjectiveThe recurrence and metastasis of nasopharyngeal cancer (NPC) may be mainly attributed to the persistence of cancer stem cells (CSCs); however, the linkage mechanism has yet to be fully elucidated.MethodsThe levels of miR-4721, FOXA1, and Nanog expression in NPC were detected by in situ hybridization and immunohistochemistry. In vivo and in vitro metastasis assays confirmed miR-4721 promotes cell migration and invasion. Tumor spheroid formation assay, side population (SP) assay, and ALDEFLUOR assay verified miR-4721 regulates cancer stem cell-like properties. Luciferase reporter assay showed that miR-4721 directly regulates FOXA1 and FOXA1 effects the promoter activity of miR-4721 and Nanog. Chromatin immunoprecipitation (ChIP) analysis and electrophoresis mobility shift assay (EMSA) revealed that FOXA1 combined the promoter region of human miR-4721 and Nanog and the possible mechanism was also analyzed.ResultsIn this study, a new mechanism of NPC tumorigenesis related to miR-4721 was verified. We found that miR-4721, FOXA1 and Nanog control their expressions through a negative feedback loop and then activate the downstream regulator of stem cell signaling to promote the enrichment and metastasis of NPC stem cells.ConclusionThese findings elucidate that the feedback loop of miR-4721/FOXA1/Nanog can regulate stemness and metastasis in NPC and may provide an experimental theoretical basis for metastasis and treatment resistance in NPC.

CK2α causes stemness and chemotherapy resistance in liver cancer through the Hedgehog signaling pathway

BackgroundLiver cancer is one of the major causes of cancer-related deaths globally. Cancer cell stemness and chemotherapy resistance contribute to the high mortality. Although evidence indicates that the alpha subunit of protein kinase 2 (CK2α) is involved in several human cancers, its function in liver cancer remains unknown. In the present study, we aimed to elucidate the role of CK2α in liver cancer. MethodsWe examined the role of CK2α regulation in stemness and chemotherapy resistance capacity of liver cancer cells. MTT assays, tumor sphere formation assays, RT-PCR, flow cytometry, Western blotting assay, clonogenicity assay, matrigel invasion assay and bioinformatics were conducted in this study. ResultsCK2α expression in the liver cancer tissues was notably upregulated compared with that in the corresponding non-tumorous tissues. The overexpression of CK2α promoted tumor sphere formation, increased the percentage of CD133(+) and side population cells, caused the resistance of liver cancer cells to 5-FU treatment, increased the expression levels of NANOG, OCT4, SOX2, Gli1 and Ptch1, and enhanced the ability of CD133(+) cell clone formation and invasion. Consistently, the downregulation of CK2α had the opposite effects. CK2α silencing inhibited the Hedgehog pathway by reducing the expression of Gli1 and Ptch1. Mechanistically, CK2α regulation on liver cancer cell stemness and chemotherapy resistance was found to be involved in the Hedgehog signaling pathway. ConclusionsOur study may bring some new insights into the occurrence of liver cancer. Furthermore, these findings suggest that targeting CK2α may be a novel therapeutic strategy for patients with liver cancer.

Butein induces intrinsic pathway of apoptosis, vimentin proteolysis, and inhibition of cancer stem cell population in a human papillary thyroid cancer cell line

Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) play an essential role in metastasis of papillary thyroid cancer (PTC). Further mesenchymal marker vimentin is linked with metastasis and cancer stem cell generation. Hence, inhibition of EMT and effective elimination of CSCs offers a novel target for the development of new therapeutic agents.The present study observed that at lower concentration, butein, a major bioactive chalcone, significantly inhibits NPA (papillary thyroid cancer cell line) cell migration and reduces extracellular acidification rate (ECAR) an indicator of enhanced glycolysis, required for cell migration. Additionally, at lower concentrations, butein treatment also suppresses vimentin phosphorylation, an essential step in cell migration, proving its potential against cell migration. Phosphorylation of vimentin is crucial in the protection of vimentin from caspase-mediated proteolysis. Interestingly, butein activates caspase-3 for the apoptosis execution at higher concentration; hence, total levels of vimentin were investigated. Butein induces caspase-3 mediated proteolysis of vimentin. Vimentin and glycolysis are essential for maintaining CSCs; therefore, aldeflour assay and side population assay were performed to investigate the effect of butein on CSCs. Our data suggest butein mediates the reduction in CSCs population.Here we report a novel mechanism of butein mediated inhibition of NPA cells migration by suppressing vimentin phosphorylation and its subsequent proteolysis. Collectively our data suggest the potential of butein as an innovative anticancer therapeutic agent for PTC management.

Correction: Downregulation of MicroRNA-644a Promotes Esophageal Squamous Cell Carcinoma Aggressiveness and Stem Cell-like Phenotype via Dysregulation of PITX2.

In the original version of this article (1), the authors found five images that were published in error: Fig. 2B, colony formation assay of KYSE-140/miR-644a; Fig. 2C, migration assay of KYSE-140/control (0h); Fig. 2D, invasion assay of KYSE-140/miR-644a; Fig. 4C, “Scramble” proportion of side population cells (top image); and Fig. 5C, alpha-tubulin Western blots (right image). The errors have been corrected in the latest online HTML and PDF versions of the article. The authors regret the errors.

O-065 The naughty cells of the endometriumxx

Abstract Stem/progenitor cells are the naughty cells of the endometrium! The term “naughty” has a number of connotations, one being immaturity which I will apply to the rare stem/progenitor cell populations hiding in the endometrium, where they have eluded scientists for so long. Despite their rarity, these immature cells have the capability of growing up and differentiating into the functional cells of the endometrium, producing their progenies in the process. The self-willed human endometrial epithelial progenitor cells (eEPC) and mesenchymal stem cells (eMSC) first revealed themselves through their clonogenic activity, shunning their mates and setting up clones of cells on their own. Their risqué production of identical copies of themselves ensures their continuity, much to the chagrin of their mature counterparts. They are sneaky and can produce large numbers of mature progeny, but rarely proliferate themselves preferring to take life easy and do little. They also spit out viability dyes (Hoechst) at a greater rate than mature endometrial cells to become Side Population (SP) cells. A number of approaches have been used to tame these naughty endometrial stem/progenitors. In order to determine the identity and location of these elusive cells, specific markers had to be found. The immature endometrial epithelial progenitor cells play tricks with the specific markers they express. For example, clonogenic eEPC are N-cadherin+, an epithelial mesenchymal transition marker, found by unbiassed gene profiling, revealed their hiding place in the bases of glands deep in the endometrial basalis. Similarly, SSEA-1+ basalis epithelial progenitors pirated their marker from mature neutrophils and differentiating human pluripotent stem cells. In mice the stem/progenitor cells like to play chase, with lineage tracing of individual genetically marked cells revealing their location in the intersection zone of the glands and luminal epithelium, and also in the gland bases (Axin2+ and Lgr5+). The identity of eMSCs has also been determined by discovery of specific markers, but even here the eMSC play games in human endometrium where sometimes they are pericytes (CD140b and CD146 double positive cells), sometimes perivascular cells (SUSD2+) and sometimes CD34+ cells in the adventitia of blood vessels. They are also adventitial perivascular cells in ovine endometrium, but this time they are CD271+. Mature endometrial stromal cell progeny are also naughty, often pretending to be eMSC, particularly when shed into menstrual fluid, confusing many of their status. Adding further to their misbehaviour, they express the same official MSC surface markers. To get even immature endometrial MSC strike back, claiming immunomodulatory properties in attempt to upstage their mature stromal progeny, also endowed with these properties. Finally, other endometrial cells such as macrophages may also be naughty as their mischievousness in evading detection can trick us to consider them as stem cells from the bone marrow, masquerading as endometrial epithelial or stromal cells. Naughty implies behaving badly and I will show data suggesting that stem/progenitor cells may escape the endometrium to cause a nasty disease, endometriosis. They may also become wayward and unruly, invading the myometrium to form adenomyosis. Some naughty epithelial progenitors defiantly pick up mutations to become cancer stem cells and initiate endometrial cancer. They may also malfunction because they do not obey estrogen signalling instructions, failing to proliferate and causing thin unresponsive endometrium. In their naughtiness, they may run away or get totally lost, thereby resulting in Asherman’s syndrome. Therefore, for numerous reasons, stem/progenitor cells are the naughty cells of the endometrium. © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Adaptation of flocculent anammox culture to low temperature by cold shock: long-term response of the microbial population

ABSTRACT Partial nitritation-anammox (PN/A) process will substantially reduce the costs for the removal of nitrogen in the mainstream of municipal sewage. However, one of the mainstream PN/A challenges is to reduce the time necessary for the adaptation of anammox bacteria to lower temperatures in mild climates. In this study, we exposed anammox flocculent culture to cold shocks [35°C → 5°C (8 h) → 15°C] and evaluated long-term cold shock response. Over a post-shock period of 40 d at 15°C, the nitrogen removal rates in the shocked culture were significantly higher compared to control, with maximum rates up to 0.082 and 0.033 kg-N/kg-VSS/d or 0.164 and 0.076 kg-N/m3/d, for shocked culture and control, respectively. In the corresponding semi-batch cycles, the shocked culture was on average 136 ± 101% more active than the control, due to the negative effect of cold shock on side populations and more active anammox cells. Per FISH, Ca. Brocadia anammoxidans and Ca. Scalindua survived the shock and remained present throughout. Thus, both anammox microorganisms seem to respond favourably to cold shocks. In sum, we provide further evidence that cold shocks accelerate the adaptation of anammox to the mainstream of municipal WWTPs. Further, for the first time, we report the long-term adaptive response of anammox to cold shocks.

Abstract 1406: FOXC1 induces cisplatin resistance through enhancer activation in bladder cancer

Abstract Introduction: We previously reported that an aggressive subpopulation of highly tumorigenic, drug-resistant bladder cancer cells can arise from the bulk tumor cells without mutational events, and that this phenotypic plasticity is driven at least in part by epigenetic mechanisms. In the current work, we analyzed the chromatin accessibility of enhancers to identify transcription factors that contribute to this transition to a drug-resistant state. Materials and methods: We used Hoechst nuclear staining and fluorescence activated cell sorting to isolate the Hoechst-low drug resistant side population (SP) cells from the less drug resistant Hoechst-high non-side population (NSP) cells in bladder cancer cell lines. We performed RNA sequencing and AcceSssIble assay to profile RNA expression, global DNA methylation and chromatin accessibility in SP and NSP cells. Putative enhancer regions were identified using H3K27ac ChIPseq data in bladder cancer cell lines, which are publicly available. We next performed transcription factor motif analysis on accessible enhancers near genes overexpressed in SP cells relative to NSP cells. The differentially expressed candidate target transcription factor by RNA-seq was validated using qRT-PCR. Functional validation was done using CRISPR knock out and drug resistance assays. Results: A higher percentage of enhancers were differentially accessible between SP and NSP cells compared with the promoters. We identified 746 enhancers that are accessible only in SP cells and 465 enhancers only in NSP cells. Transcription factor motif analysis showed that FOX family motif was enriched in accessible enhancers near SP-overexpressed genes. Among FOX family transcription factors, FOXC1 was the only overexpressed transcription factor, and it was also the most significantly overexpressed gene in SP cells. FOXC1 was consistently overexpressed in bladder tumor tissues relative to normal bladder tissues in three independent studies in BaseSpace Correlation Engine. CRISPR knock-out of FOXC1 in multiple bladder cancer cell lines resulted in decreased cisplatin resistance. Conclusion: We found that FOXC1 is overexpressed and its motif is enriched on the accessible enhancers when bladder cancer cells shift to a drug-resistant tumorigenic phenotype. Conversely, FOXC1 knock-out attenuates cisplatin resistance in bladder cancer cells. These observations suggest that differential expression and enhancer binding of FOXC1 promotes the previously observed, mutation-independent shift towards cisplatin resistance in bladder cancer. Citation Format: Yi-Tsung Lu, Tong Xu, Tien-Chan Hsieh, Maheen Iqbal, Zhifei Luo, Peggy J. Farnham, Suhn K. Rhie, Amir Goldkorn. FOXC1 induces cisplatin resistance through enhancer activation in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1406.

Abstract LB131: Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells

Abstract Aim: We investigated the mechanisms driving the concomitant overexpression of ABCG2 and CD133 in cancer stem cells (CSCs) and examined the effect of modulating the underlying machinery on anticancer drug response. Background: Multidrug resistance is a major obstacle to chemotherapy. It is usually associated with increased efflux of anticancer drugs by ATP-binding cassette transporters including ABCG2. ABCG2 is also a key survival factor for CSCs, which lead to recurrence. CSCs have been identified in patient tumor specimens and in cancer cell lines. The CSC hypothesis suggests that anticancer therapies have to target and destroy all residual CSCs in order to produce a durable response. CD133 is a well-characterized stem cell biomarker and it plays crucial role in the self-renewal and tumorigenesis of CSCs. Importantly, ABCG2 and CD133 are co-expressed in CSCs, yet the mechanism(s) driving their concomitant high expression remain elusive. mRNA polyadenylation is a key step regulating the maturation of eukaryotic mRNAs by adding a poly A tail to their 3' ends. Alternative polyadenylation, commonly observed in cancer cells, is a phenomenon responsible for the production of multiple mRNA isoforms bearing different length of 3'-untranslated region (3'UTR). Method: Sub-population of cancer cells from three ABCG2-overexpressing multidrug resistant colon cancer cell lines were sorted by Hoechst dye exclusion or CSC markers (DCLK1+LGR5+BMI1+), respectively, as side population (SP) and putative CSC cells. Expression of ABCG2, CD133, and a few master mRNA polyadenylation trans-acting regulators was compared in SP (or CSC) versus non-SP (or non-CSC) cells. Altered 3'UTR isoforms of ABCG2 and CD133 were examined by 3'RACE assay and 3'UTR isoform specific PCR. The anticancer activity of common chemotherapeutic drugs was examined after genetic modulation of the key mRNA polyadenylation regulators identified. Result: The mRNA stability and expression of both ABCG2 and CD133 were found to be remarkably higher in SP cells (or CSC cells) than in non-SP cells (or non-CSC cells). By 3'RACE assay, shortening of ABCG2 and CD133 3'UTR was evidenced in SP (and CSC) cells but not in non-SP (and non-CSC) cells, thereby increasing mRNA stability and/or translation by escaping the repression from regulatory elements found on long mRNA 3'UTR. Expression of two master mRNA polyadenylation regulators (PABPN1 and CSTF2) was found to be remarkably lower and higher, respectively, in SP (and CSC) than in non-SP (and non-CSC) cells, presumably allowing the preferential utilization of proximal 3'UTR to mediate high expression of ABCG2 and CD133. Genetic silencing of CSTF2 was shown to enhance the anticancer effect of SN-38 and reduce the colony formation capacity of CSC cells. Conclusion: A novel mRNA 3'UTR shortening machinery was shown to mediate the simultaneous overexpression of ABCG2 and CD133 in SP and CSC cells. It may represent useful drug target for circumvention of resistance and eradication of CSCs. Citation Format: Kenneth K.W. To. Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB131.

Identification of the Side Population Associated with ATP-Binding Cassette Transporters Activity Using Imaging Flow Cytometry

Identification of the Side Population Associated with ATP-Binding Cassette Transporters Activity Using Imaging Flow Cytometry

Identification of a Patients-Derived CD34+ Myeloma Cell Population with Tumor-Propagating Capacity and Resistance to Anticancer Drugs

Novel therapeutic agents have substantially improved the clinical outcomes of multiple myeloma (MM), although the majority of MM patients eventually relapse and ultimately die from therapy-refractory disease. Early studies documented the presence of clonogenic populations with MM stem-like properties in MM patients, which presumably arise from post-germinal center B (GCB) cells and are resistant to current therapies. However, both biologic properties and their ability of these cells have remained unclear. In this study, we attempted to isolate these cells from phenotypically defined (CD38+138+19-45-) MM cells (hereafter PhMMs) in bone marrow (BM) and characterized them.BM samples were obtained from 48 MM patients, including 26 newly diagnosed (NDMM) and 22 with relapsed or refractory disease (RRMM) according to the protocol approved by the Institutional Review Board of the Kindai University Faculty of Medicine. Since the side population (SP) phenotype has been considered to represent the stem cell population in various types of normal and tumor tissues, we first analyzed the expression of HSC/HPCs markers (e.g. CD34, 49f, 90, 133) in the PhMMs SP population. Among the HSC/HPCs markers, the expression levels of CD34 was significant higher in SP cells compared with those in the main population (MP) cells (mean±SD, MFI of SP vs MP in PhMMs: 925.0±84.5 vs 29.2±4.2, n=5, p<0.01). The median count of CD34+ cells in PhMMs was 0.63% (interquartile range, 0.20), and a large number of these CD34+ PhMMs displayed the same Ig light chain with the unseparated myeloma cell population. Furthermore, FISH analyses on these cell populations confirmed the presence of cytogenetic abnormalities, such as t(11;14) and del(17p) in almost examined cells, while these abnormalities were not observed in the CD34+38-45dim HSC/HPCs subsets of the same patient, suggesting that CD34+ PhMMs contain clonotypic cells with genetic abnormalities. Next, we investigated the % of CD34+ PhMMs at various disease status. Notably, the % of CD34+ PhMMs was significantly higher in RRMM than in NDMM samples. Also, paired analysis showed that the % of CD34+ fraction was significantly higher after therapy than before therapy in PhMMs, indicating that CD34+ PhMMs are more resistant to anticancer drugs than CD34- PhMMs.Next, we performed a colony forming cell assay by platting CD34+ and CD34- PhMMs isolated from 12 patients (2.5×103/dish), respectively. While CD34- PhMMs developed no colony, CD34+ PhMMs vigorously generated many large colonies in all cases, demonstrating that CD34+ PhMMs but not CD34- PhMMs have strong clonogenic and proliferative capacities. When CD34+ and CD34- PhMMs from 4 MM patients were injected intravenously into sublethally irradiated NOD/shi-scid, IL-2RγKO Jic (NOG) mice (6.0×103-1.0×104 cells/mice), CD34+ PhMMs from all 4 patients were fund to be engrafted in the BM by 24 weeks after transplant, while none of the mice transplanted with CD34- PhMMs showed no engraftment by the same time point. Importantly, all engrafted cells from CD34+ PhMMs had the same Ig light chain with patient myeloma cells. Interestingly, both CD34+ and CD34- PhMMs were identified in BM of mice receiving CD34+ PhMMs. These results indicate that myeloma-propagating activity in PhMMs can be distinguished based on the CD34 expression. To gain insights into the cell origin of CD34+ PhMMs, we quantify the expression levels of selected markers associated with the development of B cell (ex, GATA2, PU1, EBF1, OCT2, BCL6, PAX5, IRF4, BLIMP1, and XBP1) by qRT-PCR analysis using purified CD34+ and CD34- PhMM pairs from 6 patients. Gene expression profiling revealed that CD34- PhMMs had a plasma cell signature (down-regulation of Pax5, and up-regulation of IRF4, BLIMP1, and XBP1), whereas CD34+ PhMMs demonstrated a more immature pre-GCB signature(down-regulation of BCL6, IRF4, and XBP1), except for the aberrant suppression of PAX5 and the expression of GATA2 .In conclusion, this study revealed that phenotypically restricted cell population (PhMMs) contains a minor CD34+ myeloma cell fraction that originate from pre-GCB cells and possess myeloma-propagating activities, as well as resistance to anticancer drugs. Thus, the identification of these clonogenic MM propagating cells provides a basis for better understanding the pathogenesis of MM and for designing novel therapeutic strategies aimed to eradicate total MM cells. DisclosuresNo relevant conflicts of interest to declare.

Novel Stem Cell-Targeted Therapy for Multiple Myeloma Based on Dual Inhibition of EZH1/2

Multiple myeloma (MM) is largely incurable because relapse eventually occurs, despite the recent development of novel therapies. This is mainly caused by a remaining population of drug-resistant myeloma stem cells. There is a side population (SP) enriched with myeloma stem cells. Therefore, targeting SP cells may be a promising strategy to prevent and treat MM relapse.Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important epigenetic regulators that maintain the “stemness” of embryonic and hematopoietic stem cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) are catalytic components of PRC2, which trimethylate histone H3 at lysine 27 (H3K27me3) to repress transcription of target genes. Mutation and overexpression of EZH2 are associated with many cancers, including MM. Here, we found that expression of EZH1/2 was significantly higher in SP cells than in non-SP cells. These results suggest that overexpression of EZH1/2 is important for maintaining the stemness of MM cells and that both EZH1 and EZH2 are potential therapeutic targets.We developed a novel EZH1/2 dual inhibitor, OR-S1, which potently inhibited both EZH1 and EZH2, and used it to investigate the effect of pharmacologic inhibition of EZH1/2 on MM. OR-S1 suppressed the proliferation of all MM cell lines tested, and its GI50 value was significantly lower than that of the EZH2-specific inhibitor GSK126. Furthermore, flow cytometric analysis revealed that treatment with OR-S1 significantly depleted the SP cells. These results suggest that dual inhibition of EZH1/2 using OR-S1 eradicates myeloma stem cells.RNA sequencing analysis revealed that the transcriptional profiles of OR-S1-treated MM cell lines were characterized by up-regulation of genes related to the Wnt pathway. RT-PCR confirmed that OR-S1 treatment markedly increased expression of Wnt and Frizzled family members. Furthermore, western blot analysis revealed that non-phosphorylated (active) β-catenin was strongly expressed in OR-S1-treated cells. To determine whether there is direct crosstalk between PRC2 and Wnt signaling, we performed chromatin immunoprecipitation (ChIP)-sequencing using MM cell lines. This revealed that most Wnt and Frizzled loci were marked by H3K27me3. Furthermore, ChIP-qPCR analysis demonstrated that dual inhibition of EZH1/2 using OR-S1 markedly decreased the level of H3K27me3 at most of these loci. These results demonstrate that PRC2 directly targets Wnt signaling and that dual inhibition of EZH1/2 using OR-S1 activates canonical Wnt signaling.Increased activation of the canonical Wnt signaling pathway reduces self-renewal and differentiation of hematopoietic stem cells. Therefore, we generated β-catenin-overexpressing MM cells to examine the effects of increased Wnt signaling. Surprisingly, proliferation of these cells was significantly lower than that of control cells. Furthermore, flow cytometric analysis revealed that overexpression of β-catenin significantly depleted the SP cells. These results suggest that over-activation of Wnt signaling induced by dual inhibition of EZH1/2 reduces the proliferation of MM cells by inhibiting self-renewal, which is important to maintain the stemness of MM cells.Oral administration of OR-S1 to mice bearing MM xenografts significantly impaired subcutaneous tumors. Long-term administration of OR-S1 at lower doses to mice bearing orthotopic xenografts eradicated minimal residual disease from bone marrow and completely cured MM, without eliciting any serious side effects (Figure). Furthermore, we developed an orthotopic patient-derived xenograft model using specimens from a relapsed and heavily pretreated MM patient. In this model, OR-S1 treatment reduced the serum levels of human immunoglobulins. These results indicate that EZH1/2-targeting therapy with OR-S1 is also effective against a human MM xenograft.Taken together, our results strongly suggest that dual inhibition of EZH1/2 is a promising therapeutic approach to eradicate myeloma stem cells and could lead to important advances in the treatment of MM. [Display omitted] DisclosuresNakagawa:Daiichi Sankyo Co.: Research Funding. Fujita:Daiichi Sankyo Co.: Research Funding. Honma:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo: Employment. Kitabayashi:Daiichi Sankyo Co.: Research Funding.

Humanized Anti-CD26 Monoclonal Antibody Has Anti-Myeloma Efficacy for the Treatment of High-Risk Multiple Myeloma

Background: Despite remarkable progress in treatment options of multiple myeloma (MM) over the last decade with proteasome inhibitors and immunomodulatory drugs (IMiDs), most of patients with high-risk multiple myeloma (MM) relapse and ultimately become refractory. Recently, novel monoclonal antibody directly targeting CS1(elotuzumab) or CD38 (daratuzumab) induced durable responses in patients with multidrug-refractory MM. CD26, a 110-kDa transmembrane glycoprotein which is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis, whereas its role in plasma cell malignancies has not been characterized yet. Our recent work has revealed that CD26 is intensely expressed on activated osteoclasts (OCs) in MM. In addition, in vitro studies showed that although CD26 expression is only slightly detected on MM cell lines cultured alone, it is intensely and uniformly expressed on MM cell lines co-cultured with OCs. In this study, we clarify the direct on-tumor and immune mechanism of action with huCD26mAb, a humanized monoclonal IgG1 antibody specifically targeting CD26, alone or in combination with approved agents in high-risk MM.Methods and Results: Immunostaining on bone marrow biopsy specimens showed that CD26 is expressed on plasma cells in several MM patients, but not in those of healthy individuals. So, we first analyzed the effect of huCD26mAb on survival of MM cell lines (KMS18, KMS26, KMS27, KMS28, KMS34, U266). HuCD26mAb had no direct effect on proliferation in any of tested CD26- MM cell lines cultured alone, but it inhibited growth of CD26+ MM cell lines co-cultured with OCs at higher concentrations (>10μg/ml), as measured by the MTT assay. Next, we examined the ability of huCD26mAb to lyse CD26 positive MM cell lines by antibody-dependent cellular cytotoxicity (ADCC) using the calcein-AM release assay. HuCD26mAb, but not isotype control IgG1, triggered ADCC against CD26+ KMS18, KMS26, KMS28, KMS34 and U266 with t(4;14) by natural killer (NK) effector cells in a dose-dependent manner with lytic activity starting at 0.0001μg/ml and maximum lysis at 10μg/ml and in an effector to target (E/T) ratio-dependent manner. Moreover, huCD26mAb dose-dependently induced lysis of lenalidomide-resistant MM.1R via ADCC. We further asked whether pretreatment with conventional or novel agents is able to induce superior ADCC activity by huCD26mAb. CD26+ KMS18, KMS26 and KMS28 were pretreated with bortezomib (3nM), lenamlidomide (0.5μM) or dexamethasone (25nM) overnight. Indeed, subsequent MM cell lysis triggered by huCD26mAb (10μg/ml) was significantly augumented. Treatment with lenalidomide (0.5μM) followed by huCD26mAb (10μg/ml) also markedly reduced the viable MM.1R in the presence of NK effector cells. The synergistic ADCC efficacy of huCD26mAb in combination with lenalidomide resulted in 2.0-fold increase in MM.1R lysis. In addition, pretreatment of NK effector cells with lenalidomide also potentiated ADCC against MM.1R mediated by huCD26mAb in a dose-dependent fashion. Complement dependent cytotoxicity (CDC) of CD26+ MM cells by huCD26mAb was also tested using human serum as a source of complement. However, in the presence of human serum, the incubation with huCD26mAb or isotype IgG1 exerted no effect on MM cell lysis. These results indicate that huCD26mAb (≦10μg/ml) kills CD26+ MM cells by inducing ADCC but not CDC, regardless of sensitivity or resistance to conventional or novel agents. Finally, to further determine whether huCD26mAb alone or combination with lenalidomide affects side population (SP) fractions of drug-resistant MM cells, we first examined CD26 expression in SP and main population (MP) fractions by flow cytometry. Both CD26+ RPMI8226 and KMS11 exhibited SP fractions which equally expressed CD26 at high levels as MP fractions. So, we examined ADCC activity by huCD26mAb (10μg/ml) against these SP fractions, mixed with NK effector cells. Interestingly, although lenalidomide alone did not decrease the ratio of SP fractions, huCD26mAb substantially reduced their ratio and its further reduction was observed with both in combination against CD26+ RPMI8226 and KMS11.Conclusions: HuCD26mAb induced significant anti-MM efficacy chiefly through ADCC. CD26 may be a novel valuable target for antibody therapy and huCD26mAb, alone or in combination, may be a promising immunotherapeutic strategy for the treatment of high-risk MM. DisclosuresMorimoto:Y's Therapeutics: Consultancy, Research Funding. Yamada:Y's therapeutics: Consultancy, Research Funding.

Downregulation of BMI-1 By the Small Molecule PTC596 Induces Mitochondrial Apoptosis in Mantle Cell Lymphoma

▪Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma typically associated with the t(11;14) translocation, resulting in overexpression of cyclin D1 (CCND1). Although the clinical approval of the first-in-class Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a new treatment option, the great majority of patients with MCL remain incurable. Studies are needed to optimize the role of ibrutinib in MCL therapy and to address ibrutinib resistance. BMI-1 is crucially involved in cancer stem cell self-renewal and maintenance. BMI-1 has been shown to be up-regulated in MCL cells, especially in tumor-initiating side population (SP) cells (Teshima et al. Oncogene 2014). Importantly, BMI-1 levels are higher in refractory/relapsed patients than those at initial diagnosis. PTC-209 and PTC596 are recently developed novel small-molecule selective BMI-1 inhibitors with different modes of action (Kreso et al. Nat Med 2014). PTC-209 inhibits BMI-1 production while PTC596 accelerates BMI-1 degradation. We have reported anti-leukemia effects of the BMI-1 inhibitors PTC-209 and PTC596 (Cancer Sci 2015; Blood Cancer J 2017) in acute myeloid leukemia cell lines and patient-derived leukemia blasts. In this study, we investigated the effects of PTC-209 and PTC596 in MCL. Oncomine analysis showed that BMI1 mRNA levels were higher in MCL cells than in normal B cells. The levels were not specifically associated with CCND1 levels, ATM, CDKN2A or TP53 mutations, or patient prognosis, implying that BMI1 overexpression may be a fundamental abnormality in MCL. PTC-209 and PTC596 inhibited cell growth and induced apoptosis in 8 MCL cell lines in a dose- and time-dependent manner. Their IC50 values (concentration at which cell growth is inhibited by 50%) ranged from 1.5 to 11.2 µM for PTC-209 and from 68 to 340 nM for PTC596. ED50 values (effective concentration inducing 50% cell killing as measured by annexin V positivity) ranged from 2.7 to > 50 µM for PTC-209 and from 150 to 507 nM for PTC596. PTC596 was > 10 times more potent than PTC-209. IC50 and ED50 values of PTC-209 significantly correlated with those of PTC596 [r =0.94 (P= 0.0004) for IC50 and r =0.85 (P= 0.015) for ED50], supporting the idea that their anti-lymphoma activities depend on downregulation of BMI-1. In support of this, high BMI-1 protein levels predicted high susceptibility to PTC596 (r = -0.88; P=0.0039). The anti-lymphoma effects of PTC596 appeared to be p53-independent since IC50 and ED50 values were not significantly different against p53 wild-type and p53 defective cell lines. In addition, stable knockdown of p53 did not significantly affect MCL cell susceptibility to PTC596. Activity of PTC596 was independent of MCL cell susceptibility to ibrutinib. PTC596 induced mitochondrial apoptosis, as evidenced by loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation and phosphatidylserine externalization in MCL cells. PTC596 effectively decreased SP cells (by 97.3% at 150 nM, 99.8% at 300 nM and >99.9% at 600 nM PTC596) compared to ibrutinib that decreased SP cells by 33% even at 5 µM ibrutinib. Combination of PTC596 with bortezomib or doxorubicin showed additive anti-proliferative and apoptotic effects. The averaged combination index values were approximately 1, ranging from 0.95 to 1.18. Interestingly, PTC596 synergized with ibrutinib in inducing cell death, through modulation of the BCL-2 family protein MCL-1. Collectively, BMI-1 inhibition may offer a novel therapeutic strategy for MCL. DisclosuresLiangl:PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment. Branstrom:PTC Therapeutics: Employment.

Regulatory T Cells Promote the Stemness of Acute Myeloid Leukemia Cells through IL10 Cytokine Related Signaling Pathway

Regulatory T cells (Tregs) could maintain the stemness property and inhibit the differentiation of hematopoietic stem/progenitor cells (HSPCs) in normal hematopoietic system. Recent studies have shown that Tregs, as an important component of acute myeloid leukemia (AML) microenvironment, helped AML cells to evade immunoesurveillance. However their function in regulating the stemness property of AML cells remains elusive.In this study, we analyzed the proportion of Tregs and leukemic stem cells (LSCs) in 68 patients' bone marrow, and found that the proportion of LSCs in Tregs high group (Tregs ≥ 9%) was higher than that in Tregs low group (Tregs < 9%) (Figure A). So, we hypothesized that Tregs up-regulated the stemness property of AML cells. Therefore, we treated Kasumi-1 and SKNO-1 with the supernatant of Tregs culture medium (condition medium, CM) which was isolated from donors, and verified that the proportion of side population (SP) cells was increased (Figure B) and the efficiency of sphere formation was enhanced (Figure C) by CM treatment. Further, Tregs promoted the initiation of AML and shortened the life span of mice in AML1-ETO mice model (Figure D).As is well known that, Tregs could secrete IL10 cytokine to establish a suppressive microenvironment. Therefore, we analyzed the expression level of IL10 in 35 patients' bone marrow, and found that the expression of IL10 in Tregs high group was higher than that in Tregs low group (Figure E), meanwhile, the expression of IL10 in LSCs high group (LSCs ≥ 5%) was higher than that in LSCs low group (LSCs < 5%). Thus, we treated Kasumi-1 and SKNO-1 with human recombinant IL10 cytokine (rhIL10). rhIL10 treatment increased proportion of SP, enhanced the efficiency of sphere formation and induced both activating phosphorylation of PI3K and AKT at S473 with similar kinetics in a dose-dependent (with concentrations ranging from 1 to 100 nM) manner. Given the ability of AKT to increase the expression of OCT4 and nanog, we analyzed changes in the expression of OCT4 and nanog. As expected, expression levels of OCT4 and nanog increased after treatment with IL10.To investigate whether IL10 is responsible for stemness property induction, we blocked PI3K/AKT signaling pathway by IL10R neutralizing antibody (IL10R Ab) and PI3K/AKT inhibitor- LY294002 following CM treatment. In contrast to the control (CM treatment only), LY294002 and IL10R Ab could greatly reduce the proportion of SP, lessen the efficiency of sphere formation, and decrease the expression of OCT4 and nanog.Consequently, our study revealed that Tregs increased the proportion of LSCs in AML through IL10/PI3K/AKT/OCT4/nanog signaling pathway and indicated that targeting on the communication between Tregs and AML cells might be a promising strategy in AML therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CircPVT1 Inhibit Apoptosis and Enhance Drug Resistance in Multiple Myeloma

Objective: Circular RNAs (circRNAs) comprise a novel class of widespread non-coding RNAs that could regulate gene expression in eukaryotes. Glucocorticoid resistance is the primary factor of refractory relapse in patients with multiple myeloma (MM). This study was designed to reveal the role of circPVT1 in mediating glucocorticoid resistance in MM through regulating its target genes to inhibit apoptosis of MM cell and promote tumor stem cell survival.Methods and results: We determined with RT-PCR assay that circRNA PVT1 is differentially expressed between glucocorticoid resistant and sensitive MM cells (Figure A), and is closely correlated with prognosis (Figure G and H). We further investigated the role of circPVT1 in MM cell apoptosis and proliferation as well as its downstream signaling pathway mechanism. Silencing circPVT1 enhances sensitivity to glucocorticoid treatment (Figure D), promotes apoptosis and inhibits cell proliferation in resistant MM cell lines and myeloma cells in NOD-SCID mice, resulting up-regulation of the apoptosis pathway as measured by increased Caspase3, PARP and decreased Bcl-2 (Figure E). Measured by CCK-8 cell viability assay, ectopic expression of circPVT1 contributed to inducing proliferation and suppressing apoptosis in sensitive MM cell lines. Using Hoechst Staining, we found that MM stem cell population increases upon ectopic expression and decreases while silencing circPVT1, which revealed circPVT1 could facilitate the proliferation of stem cells to enhance glucocorticoid resistance (Figures F and I).Conclusion: As a downstream of circPVT1, it promotes c-myc nuclear translocation, which intermediated by miRNAs to enhance its targets thus promotes the proliferation of stem cells and inhibits apoptosis. We concluded that circPVT1 may mediate MM glucocorticoid resistance and its downstream signaling pathway may represent a novel target for MM treatment.Figure (A)RT-PCR showing the differential expression of circPVT1 and linear RNA PVT1 between MM.1R and MM.1S cell lines.** P <0.01. Error bars, S.D. (n=3). (B)RT-qPCR results showing the abundance of circPVT1 and PVT1 in MM.1R cells treated with RNase R. The levels of CircPVT1 and PVT1 were normalized to the values measured after mock treatments.** P <0.01. Error bars, S.D. (n=3). (C)The expression of circPVT1 was detected by RT-PCR and was validated by Sanger sequencing. (D)RT-PCR analysis of circPVT1 expression after treatment with siRNA targeting at the circPVT1.** P <0.01. Error bars, S.D. (n=3). (E)Western blot analysis of the levels of PARP, caspase 3, Bcl-2, control of β-actin in MM.1R and MM.1S transfected with circPVT1 siRNA or control siRNA, in absence of dexamethasone 1μM/L for 24 hours. (F)Using Hoechst Staining, analysis of side population of MM.1R transfected with circPVT1 siRNA or control siRNA. (G)RT-PCR showing the differential expression of circPVT1 of GC sensitive and resistant patients. ** P <0.01. Error bars, S.D. (n=28). (H)Kaplan-Meier analysis of the correlation between GC sensitivity and overall survival. (I)Diagram showing c-myc nuclear translocation facilitated by circPVT1, intermediated by miRNAs to enhance the targets of c-myc thus promotes the proliferation of stem cells and inhibits apoptosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.