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The current treatment paradigm of abdominal aortic aneurysms (AAA) focuses on observing patients until their disease reaches certain thresholds for intervention, with no preceding treatment available. There is an opportunity to develop novel therapies to prevent further aneurysmal growth and decrease the risk of a highly morbid rupture. We used a porcine model of aortic dilation to assess the ability of human adipose-derived mesenchymal stem cells (MSCs) to attenuate aortic dilation.Twelve Yorkshire pigs received periadventitial injections (collagenase and elastase) into a 4-cm segment of infrarenal aorta. Animals were treated with either 1 × 10All animals survived until POD 21. The mean aortic diameter was reduced in the aortic dilation + MSC treatment group compared to aortic dilation control animals (1.10 ± 0.126 versus 1.48 cm ± 0.151, P 0.001). Aortic media thickness was reduced in the aortic dilation group compared to the aortic dilation + MSC group (609.14 IQR 445.21-692.93 μm versus 643.55 IQR 560.91-733.88 μm, P = 0.0048). There was a significant decrease in the content of collagen and alpha-smooth muscle actin and elastin perturbation in the aortic dilation group as compared to the aortic dilation + MSC group. Immunohistochemistry demonstrated an increased level of vascular endothelial growth factor, tissue inhibitor of matrix metalloproteinase 1, and tissue inhibitor of matrix metalloproteinase 3 expression in the aorta of aortic dilation + MSC animals.Stem cell therapy suppressed the aortic dilation in a porcine model. Animals from the aortic dilation group showed more diseased gross features, histologic changes, and biochemical properties of the aorta compared to that of the aortic dilation + MSC treated animals. This novel finding should prompt further investigation into translatable drug and cell therapies for aneurysmal disease.

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) are crucial for maintaining lifelong hematopoiesis. Developing methods to distinguish stem cells from other progenitors and evaluate stem cell functions has been a central task in stem cell research. Deep learning has been demonstrated as a powerful tool in cell image analysis and classification. In this study, we explored the possibility of using deep learning to differentiate HSCs and MPPs based on their light microscopy (DIC) images. After extensive training and validation with large image data sets, we successfully develop a three-class classifier (we named it the LSM model) that reliably differentiate long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and MPPs. Importantly, we demonstrated that our LSM model achieved its differentiating capability by learning the intrinsic morphological features from cell images. Furthermore, we showed that the performance of our LSM model was not affected by how these cells were identified and isolated, i.e., sorted by surface markers or intracellular GFP markers. Prospective identification of HSCs and MPPs in Evi1GFPtransgenic mice by LSM model suggested that the cells with the highest GFP expression were LT-HSCs, and this prediction was substantiated later by a long-term competitive reconstitution assay. Moreover, based on DIC image data sets, we also successfully built another two-class classifier that can effectively distinguish aged HSCs from young HSCs, which both express the same surface markers but are functionally different. This finding is of particular interest since it may provide a novel quick and efficient approach, obviating the need for a time-consuming transplantation experiment, to evaluate the functional states of HSCs. Together, our study provides evidence for the first time that HSCs and MPPs can be differentiated by deep learning based on cell morphology. This novel and robust deep learning-based platform will provide a basis for the future development of a new generation stem cell identification and separation system. It may also provide new insight into molecular mechanisms underlying the self-renewal feature of stem cells.

Extracellular vesicles (EVs) are a varied group of cell-derived, microscopic, fluid-filled pouches released from cells into neighboring microenvironments that are quickly gaining recognition as a potentially powerful tool against epithelial ovarian cancer (EOC). Recent studies show that not only do EVs play an integral part in the development of cancer through intercellular communication, cell survival, and immune modulation but also may assist with early diagnosis and improved treatments. EOC currently has few effective screening options for early detection of this disease; and, therefore, it is detected at an advanced stage where it is more likely to recur, develop chemoresistance, and ultimately become fatal. Newer research has evaluated EVs as biomarkers for early screening and diagnosis and as novel targets for treatment of EOC. Moreover, EVs are possible targets for novel immunomodulatory therapies to directly target cancer cells or make cancer cells more susceptible to other treatment modalities. Therefore, EVs present an exciting, promising approach which may improve clinical outcome for EOC patients.

Introduction: The current treatment paradigm of abdominal aortic aneurysms (AAA) focuses on observing patients until their disease reaches certain thresholds for intervention, with no preceding treatment available. There is an opportunity to develop drug or cell-based therapies to prevent further aneurysmal growth and decrease the risk of a highly morbid rupture. A previously validated acute AAA swine model was treated directly with gelfoam containing human adipose-derived stem cells (h-ADSCs). Methods: Male Yorkshire swine (n=16) received periadventitial injections (type 1 collagenase and porcine pancreatic elastase) into a 4 cm segment of infrarenal aorta. Experimental group (n=7) received gel foam soaked with 10 million h-ADSCs and control group (n=9) media-soaked gel foam. Aortic diameters (AD) were monitored at post-operative day (POD) 7 and 14 using ultrasound with euthanasia on POD 21. Suprarenal (control) and infrarenal aortas (experimental) were harvested for PCR, mechanical tensiometry testing, histopathological analyses as well as immunohistochemistry (IHC). Each animal served as their own control. Groups were compared with paired t-tests and Mann Whitney U tests. Results: Twelve animals survived until POD 21. Stem cell treated AAAs, unlike controls, demonstrated no growth (-1.6 ± 8.8% vs. 34.8 ± 13.7%, p Conclusion: Stem cell therapy prevents the development of aortic aneurysms in a swine model. Stem cell treated swine with induced AAAs show more normal gross features, histologic, and biochemical properties compared to untreated controls with induced AAAs. Further investigation is merited as a possible translatable therapy for aneurysmal disease.

The HPSE gene encodes heparanase (HPSE), a key player in cancer, inflammation, and autoimmunity. We have previously identified a strong HPSE gene enhancer involved in self-regulation of heparanase by negative feedback exerted in a functional rs4693608 single-nucleotide polymorphism (SNP) dependent manner. In the present study, we analyzed the HPSE gene insulator region, located in intron 9 and containing rs4426765, rs28649799, and rs4364254 SNPs. Our results indicate that this region exhibits HPSE regulatory activity. SNP substitutions lead to modulation of a unique DNA-protein complex that affects insulator activity. Analysis of interactions between enhancer and insulator SNPs revealed that rs4693608 has a major effect on HPSE expression and the risk of post-transplantation acute graft versus host disease (GVHD). The C alleles of insulator SNPs rs4364254 and rs4426765 modify the activity of the HPSE enhancer, resulting in altered HPSE expression and increased risk of acute GVHD. Moreover, rs4426765 correlated with HPSE expression in activated mononuclear cells, as well as with CD3 levels and lymphocyte counts following G-CSF mobilization. rs4363084 and rs28649799 were found to be associated with CD34+ levels. Our study provides new insight into the mechanism of HPSE gene regulation and its impact on normal and pathological processes in the hematopoietic system.

Early diagnosis and intervention are some of the longstanding challenges associated with ovarian cancer, which is the leading cause of gynecologic cancer mortality. While the majority of patients who present with advanced stage disease at time of diagnosis will initially respond to traditional combination platinum and taxane-based chemotherapy in conjunction with cytoreductive surgery, approximately 70% will ultimately recur due to chemoresistance within the first two years. Intratumor heterogeneity is proposed to be a leading factor in the development of chemoresistance and resultant poorer outcomes for those with recurrent or advanced stage disease. Both inherent and acquired mechanisms of chemoresistance are postulated to be a result of alterations in gene expression, also known as epigenetic modifications. Therefore, epigenetic therapy is a pivotal avenue which allows for reversal of chemoresistance in cancer through the targeting of aberrant mutations. In this chapter, we discuss how these epigenetic modifications prove to be promising targets in cancer therapy leading to heightened drug sensitivity and improved patient survival outcomes.

The success of cannabinoids with chronic neuropathic pain and anxiety has been demonstrated in a multitude of studies. With the high availability of a non-intoxicating compound, cannabidiol (CBD), an over-the-counter medication, has generated heightened interest in its use in the field of oncology. This review focuses on the widespread therapeutic potential of CBD with regard to enhanced wound healing, lowered toxicity profiles of chemotherapeutics, and augmented antitumorigenic effects. The current literature is sparse with regard to determining the clinically relevant concentrations of CBD given the biphasic nature of the compound’s response. Therefore, there is an imminent need for further dose-finding studies in order to determine the optimal dose of CBD for both intermittent and regular users. We address the potential influence of regular or occasional CBD usage on therapeutic outcomes in ovarian cancer patients. Additionally, as the development of chemoresistance in ovarian cancer results in treatment failure, the potential for CBD to augment the efficacy of conventional chemotherapeutic and epigenetic drugs is a topic of significant importance. Our review is focused on the widespread therapeutic potential of CBD and whether or not a synergistic role exists in combination with epigenetic and classic chemotherapy medications.

OBJECTIVE To examine the cytotoxicity of epigenetic drugs independently and in combination with chemotherapy on ovarian cancer cells Caov-3, and to investigate their ability to acquire chemoresistance in omental microenvironments and whether epigenetic drugs can counteract this chemoresistance. METHODS A pilot study was conducted in Cooper University Hospital, NJ, USA from August 1 to October 31, 2017, among women undergoing surgeries for uterine and ovarian cancer. Cytotoxicity assays using IC50 values of epigenetic drugs and paclitaxel and cisplatin were performed on Caov-3. Omental adipose-derived stem cells (OASCs) were isolated from omentum with/without metastases. Caov-3 was cultured with OASCs' conditioned medium and subjected to different drugs. Cell viability and secretome was measured using MTT and Elisa, respectively. RESULTS Three women met the eligibility criteria and were included in the study. Epigenetic drugs alone or in combination with chemotherapy showed 85%-94% increased cytotoxicity against Caov-3 (P≤0.005). Metastatic OASCs conditioned medium showed up to 27-fold increase in tumorigenic factors and promoted chemoresistance (28%-35%; P

Contains fulltext : 232394.pdf (Publisher’s version ) (Closed access) We evaluated the association between germline genetic variants located within the 3'-untranlsated region (polymorphic 3'UTR, ie, p3UTR) of candidate genes involved in multiple myeloma (MM). We performed a case-control study within the International Multiple Myeloma rESEarch (IMMEnSE) consortium, consisting of 3056 MM patients and 1960 controls recruited from eight countries. We selected p3UTR of six genes known to act in different pathways relevant in MM pathogenesis, namely KRAS (rs12587 and rs7973623), VEGFA (rs10434), SPP1 (rs1126772), IRF4 (rs12211228) and IL10 (rs3024496). We found that IL10-rs3024496 was associated with increased risk of developing MM and with a worse overall survival of MM patients. The variant allele was assayed in a vector expressing eGFP chimerized with the IL10 3'-UTR and it was found functionally active following transfection in human myeloma cells. In this experiment, the A-allele caused a lower expression of the reporter gene and this was also in agreement with the in vivo expression of mRNA measured in whole blood as reported in the GTEx portal. Overall, these data are suggestive of an effect of the IL10-rs3024496 SNP on the regulation of IL10 mRNA expression and it could have clinical implications for better characterization of MM patients in terms of prognosis.

Objectives: High-grade serous ovarian cancer (HGSOC) is the leading cause of death from gynecological cancer in the US. The majority of patients will present at advanced stage and will recur due to chemoresistance. New therapeutic strategies such as epigenetic and cannabinoid-based therapies may help circumvent this chemoresistance problem. We aim to determine the potency of clinically relevant concentrations of CBD monotherapy as well as combined with epigenetic (ET) and classic chemotherapy on tumor formation and propagation in 3-Dimensional (3D) ovarian cancer (OC) spheroid models. Methods: We created our clinically relevant 3D spheroid tissue culture model in order to mimic physiological conditions in vivo with platinum sensitive OC cell lines. Spheroids were treated with both one time and chronic doses of cannabidiol (CBD) at pre-determined concentrations alone or in combination with chemotherapeutic and epigenetic drugs (Cisplatin/Paclitaxel, Panobinostat). Spheroid growth and morphology were evaluated over an extended time kinetic (days 0, 3, 6, 10/12) and measured by volume of growth. The average radius of the spheroids was used to calculate the spheroid volume according the formula V = 4/3 pr3 and was monitored for percent growth relative to spheroid size at the beginning of the experiment (day 0). Microscopic pictures were taken to follow the morphologic changes. Results: Over an extended kinetic, high and medium concentrations of one time CBD (CBDH) tended to stunt spheroid growth in comparison to control, (686% and over 700% less growth). Low concentrations of one time CBD (CBDL) had a lessened effect on spheroid growth, (556% less growth). Classic chemotherapy combined with CBD contributed to a decreased overall spheroid growth, and even shrinkage with one time CBDL (34% shrinkage versus 84% growth in C/T group) over an extended kinetic. Chronic CBDH administration resulted in greater than 7500% less growth by day 10. Chronic CBDL in combination with C/T resulted in a 99% less growth in comparison to C/T by day 6. One time CBD administration with Panobinostat (Pano) decreased the amount of growth over a shorter kinetic (18% and 2% shrinkage for Pano/CBDL and Pano/CBDM, respectively, versus 13% growth for Pano). With the application of chronic CBD, Pano and CBD resulted in shrinkage early on and even 100% lysis of 3D OC by day 10. Download : Download high-res image (465KB) Download : Download full-size image Conclusions: As demonstrated by our results, clinically relevant concentrations of CBD possess antitumorigenic effects on OC cells. Further, our data using the combination of Panobinostat with varying concentrations of CBD leads us to hypothesize that a one-time dose of CBD is not sufficient for prolonged antitumorigenic effects, with a synergistic effect demonstrated by chronic administration of CBD. Regarding classic chemotherapy and the effect of varying concentrations of CBD, our data indicates a synergistic effect exists with low concentrations of CBD. Further studies are indicated to determine whether these synergistic effects can aid in the reversal of chemoresistance and enhance the efficacy of targeted treatments.

Stem cell therapy promotes tissue regeneration and wound healing. Efforts have been made to prime stem cells to enhance their regenerative abilities. Certain marijuana components, namely the non-psychoactive cannabidiol (CBD) and psychoactive tetrahydrocannabinol (THC), are defined as immunomodulators.9 We test whether two sources of stem cells, primed with CBD or THC, would demonstrate improved regenerative abilities. Human adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (BMDSCs), not obtained from the same individual, were treated with low (300 nM) or high (3 μM) concentration CBD. Porcine ASCs and BMDSCs were isolated from a single pig, and treated with either low or high concentrations of CBD or THC. Transwell migration and MTT proliferation assays were performed on the human ASCs and BMDSCs. Also, transwell migration assay was performed on the porcine ASCs and BMDSCs. Finally, a wound healing scratch assay in porcine primary fibroblasts (PFs) was performed, co-cultured with the cannabinoid-treated ASCs. CBD priming at low concentration induces migration by 180% (P < .01) in porcine ASCs, and by only 93% (P < .02) in porcine BMDSCs. In porcine stem cells, THC priming at low concentration induces migration by 91.6% (P < .01) in ASCs but by only 44.3% (P < .03) in BMDSCs. Compared to PFs co-cultured with untreated ASCs, PFs co-cultured with low CBD-primed ASCs had 75% faster wound closure at 18 hours (P < .01). CBD and THC priming of ASCs and BMDSCs, particularly at lower doses, enhances a number of regenerative parameters, suggesting that these major marijuana components may improve stem cell-based therapies. SIGNIFICANCE OF THE STUDY: Our study demonstrates that cannabinoids can enhance the regenerative capacity of two major sources of stem cells, adipose- and bone marrow-derived, from human and porcine donors. Stem cell isolation and expansion is invasive, costly and time consuming. Stem cells with improved regenerative properties may be effective in the treatment of acute or chronic wounds. This is the first study to compare the priming potential of two sources of stem cells from the same animal, with the same genetic and epigenetic profile, as well as the first to prime with THC.

Ovarian cancer is uncommon in relation to other women's cancer, however, it is associated with a disproportionate number of deaths due to women's cancer. According to the National Institute of Health, only 1.2% of new cancer diagnoses in the United States are attributed to ovarian cancer, yet it is the fifth leading cause of cancer death in women and is responsible for 2.3% of all female cancer deaths. Ovarian cancer deaths are largely due to widely metastatic and chemoresistant disease that often presents at a late stage. The omentum is one of the most common sites for ovarian cancer metastasis. Recent research findings have highlighted the specific tumor microenvironment of the omentum and how it can be manipulated to prevent ovarian cancer proliferation, metastasis and chemoresistance. Debulking surgery has been the mainstay in the treatment for ovarian cancer. Total omentectomy is classically described as essential to this procedure. This article explores the known benefits of total omentectomy in the surgical treatment of epithelial ovarian cancer as well as the potential benefit contained within the omental tumor microenvironment when the omentum is macroscopically free of disease at the time of initial surgery.

Single Nucleotide Polymorphisms (SNPs) is the substitution of a single nucleotide, stably inherited, highly abundant, and distributed throughout the genome. Up today 9746 SNPs were found in the HPSE gene. During 12 years 21 SNPs were analyzed in normal and pathological samples. The most prominent SNPs are rs4693608, rs11099592, rs4693084, and rs4364254. These SNPs were found in correlation with heparanase mRNA and protein expression among healthy persons. Moreover, an association of the HPSE gene SNPs with inflammatory processes, cancer development and progression was detected. SNP investigation allowed the identification of strong HPSE gene enhancer in the intron 2. In normal leukocytes, heparanase binds to the enhancer region and regulates HPSE gene expression via negative feedback in rs4693608 SNP-dependent manner. In malignant cells, heparanase halted self-regulation of the enhancer region. Instead of heparanase, the helicase-like transcription factor (HLTF) binds to the regulatory region. These and subsequent studies will elucidate how modification in the HPSE enhancer region could be applied to develop new approaches for cancer treatment.

THC is the main psychoactive compound found in marijuana. A number of studies over the past few decades, both in vitro and in vivo, have demonstrated that THC down-regulates the inflammatory process through various mechanisms. Similar findings have been demonstrated with CBD, the other major bioactive component of marijuana. Given the essential role that inflammation plays in early wound healing, it is possible that marijuana, or its individual constituents, may impact this process. Herein, we review the existing literature related to the effects of THC on inflammation and potentially wound healing, and discuss how this connection may be relevant from a surgical perspective.

Single Nucleotide Polymorphisms (SNPs) is the substitution of a single nucleotide, stably inherited, highly abundant, and distributed throughout the genome. Up today 9746 SNPs were found in the HPSE gene. During 12 years 21 SNPs were analyzed in normal and pathological samples. The most prominent SNPs are rs4693608, rs11099592, rs4693084, and rs4364254. These SNPs were found in correlation with heparanase mRNA and protein expression among healthy persons. Moreover, an association of the HPSE gene SNPs with inflammatory processes, cancer development and progression was detected. SNP investigation allowed the identification of strong HPSE gene enhancer in the intron 2. In normal leukocytes, heparanase binds to the enhancer region and regulates HPSE gene expression via negative feedback in rs4693608 SNP-dependent manner. In malignant cells, heparanase halted self-regulation of the enhancer region. Instead of heparanase, the helicase-like transcription factor (HLTF) binds to the regulatory region. These and subsequent studies will elucidate how modification in the HPSE enhancer region could be applied to develop new approaches for cancer treatment.

Objective: (1) Determine if sequential administration of standard chemotherapy (paclitaxel and cisplatin, P/C) with epigenetic drugs effectively targets ovarian cancer and limits toxicity to normal cells. (2) Define whether epigenetic treatment can shorten the exposure to P/C. Methods: Normal cells—adipocyte-derived stem cells (ASC), primary fibroblasts (PF), and human intestinal epithelial cells (HIEC-6)—were treated with 48 h IC50 values of P/C and epigenetic drugs, 5-azacytidine (AZA) and or suberoylanilide hydroxamic acid (SAHA), in combination and sequentially. The least toxic regimens to normal cells were administered to the ovarian cancer cell lines Caov-3, SKOV-3, OVCAR-3. Cell viability after treatments were assessed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and cell count assays. Secretome analysis of conditioned medium collected from the treated ovarian cancer cells was performed using ELISA. Results: P/C with AZA and SAHA targeted all ovarian cancer cell lines (82-99% cell death), but also caused significant normal cell death (66-100%). In contrast, P/C followed by AZA or SAHA is less toxic to ASC and PF (25-96% viability) when compared to a four-drug combination therapy (1% viability, p

Heparanase is an endo-β-glucuronidase that specifically cleaves the saccharide chains of heparan sulfate (HS) proteoglycans and releases HS-bound cytokines, chemokines, and bioactive growth-promoting factors. Heparanase plays an important role in the nucleus as part of an active chromatin complex. Our previous studies revealed that rs4693608 correlates with heparanase levels and increased risk of acute and extensive chronic graft vs. host disease (GVHD). Discrepancy between recipient and donor in this SNP significantly affected the risk of acute GVHD. In the present study, we analyzed the HPSE gene region, including rs4693608, and demonstrated that this region exhibits SNPs-dependent enhancer activity. Analysis of nuclear proteins from normal leukocytes revealed their binding to DNA probe of both alleles with higher affinity to allele G. All malignant cell lines and leukemia samples disclosed a shift of the main bands in comparison to normal leukocytes. At least five additional shifted bands were bound to allele A while allele G probe was bound to only one main DNA/protein complex. Additional SNPs rs4693083, rs4693084, and rs4693609 were found in strong linkage disequilibrium (LD) with rs11099592 (exon 7). Only rs4693084 affected protein binding to DNA in cell lines and leukemia samples. As a result of the short distance between rs4693608 and rs4693084, both SNPs may be included in a common DNA/protein complex. DNA pull-down assay revealed that heparanase is involved in self-regulation by negative feedback in rs4693608-dependent manner. During carcinogenesis, heparanase self-regulation is discontinued and the helicase-like transcription factor begins to regulate this enhancer region. Altogether, our study elucidates conceivable mechanism(s) by which rs4693608 SNP regulates HPSE gene expression and the associated disease outcome.

Recurrent high-grade serous ovarian cancer (HGSC) is clinically very challenging and prematurely shortens patients’ lives. Recurrent ovarian cancer is characterized by high tumor heterogeneity, therefore, it is susceptible to epigenetic therapy in classic 2D tissue culture and rodent models. Unfortunately, this success has not translated well into clinical trials. Utilizing a 3D spheroid model over a period of weeks, we were able to compare the efficacy of classic chemotherapy and epigenetic therapy on recurrent ovarian cancer cells. Unexpectedly, in our model, a single dose of paclitaxel alone caused the exponential growth of recurrent high-grade serous epithelial ovarian cancer over a period of weeks. In contrast, this effect is not only opposite under treatment with panobinostat, but panobinostat reverses the repopulation of cancer cells following paclitaxel treatment. In our model, we also demonstrate differences in the drug-treatment sensitivity of classic chemotherapy and epigenetic therapy. Moreover, 3D-derived ovarian cancer cells demonstrate induced proliferation, migration, invasion, cancer colony formation and chemoresistance properties after just a single exposure to classic chemotherapy. To the best of our knowledge, this is the first evidence demonstrating a critical contrast between short and prolonged post-treatment outcomes following classic chemotherapy and epigenetic therapy in recurrent high-grade serous ovarian cancer in 3D culture.

Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic modalities that bypass these resistance mechanisms. FTY720, also known as fingolimod, is an S1P modulator approved by the FDA to treat the relapsing form of multiple sclerosis. Previously we reported that FTY720 exhibits potent anti-myeloma effect in vitro and in vivo in disseminated xenograft model of MM (Beider et al., Clin Cancer Res 2017). Cytotoxic activity of FTY720 was associated with down-regulation of anti-apoptotic protein MCL-1 while not affecting BCL-2 levels. It is therefore conceivable that BCL-2 inhibition using BH3-mimetic venetoclax may improve responses to FTY720. Incubation of human MM cell lines (n=8) and primary MM cells (n=3) with venetoclax and FTY720 combination synergistically potentiated cell death (CI Corresponding with mitochondrial destabilization, venetoclax/FTY720 combination promoted the release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol and subsequently increased AIF nuclear localization, suggesting its functional role in the execution phase of the apoptosis in response to the dual treatment. AIF is a mitochondrial oxidoreductase that contributes to cell death programs and participates in the assembly of the respiratory chain. Of note, single-agent treatment with FTY720 profoundly up-regulated mitochondrial AIF levels. Given the regulative role of AIF in mitochondrial bioenergetics, we could suggest that increased mitochondrial levels of AIF upon FTY720 exposure may support adaptive responses and promote MM survival upon mitochondrial stress. We thus investigated a possible effect of venetoclax and FTY720 separately or in combination on the metabolic activity of MM cells, observing distinct metabolic profiles of single versus combined exposures. FTY720 significantly suppressed glycolysis, down-regulating the transcript levels of the glycolytic enzymes HK2, PDK1, and LDHA. Glycolytic suppression may result in upregulation of mitochondrial content, which maintains cell survival. In accordance, increased mitochondrial activity was detected in FTY720-treated MM cells, detected by high uptake of MitoSpy Red, a dye that stains mitochondria in a membrane potential-dependent manner. To determine if the changes in the mitochondrial content also altered mitochondrial function, bioenergetic analysis was undertaken. FTY720-treated MM cells demonstrated increased levels of NDUFB8 and UQCRC2 (subunits of mitochondrial respiratory complexes I and III, respectively). Furthermore, FTY720 exposure up-regulated ATP production, suggesting an increase in tumor-protective oxidative phosphorylation (OXPHOS). In agreement, inhibition of mitochondrial electron transport chain using rotenone sensitized MM cells to FTY720, synergistically promoting cell death. Notably, co-treatment with venetoclax effectively reversed the metabolic changes mediated by FTY720, reducing mitochondrial mass, suppressing mitochondrial activity and strongly down-regulating the pathways related to OXPHOS. Furthermore, venetoclax/FTY720 combination significantly reduced glutathione (GSH) levels, therefore suppressing antioxidative cell responses. To conclude, we unveil venetoclax role in the metabolic regulation in MM cells. Venetoclax reverses metabolic reprogramming induced by FTY720, suppresses mitochondrial respiration, induces vigorous mitochondrial damage and preferentially targets MM cells in combination with FTY720. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Figure 1 Figure 1. Disclosures Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding.

Multiple myeloma (MM) is a malignancy of plasma cells usually infiltrating the bone marrow, associated with the production of a monoclonal immunoglobulin (M protein) which can be detected in the blood and/or urine. Multiple lines of evidence suggest that genetic factors are involved in MM pathogenesis, and several studies have identified single nucleotide polymorphisms (SNPs) associated with the susceptibility to the disease. SNPs within miRNA-binding sites in target genes (miRSNPs) may alter the strength of miRNA-mRNA interactions, thus deregulating protein expression. MiRSNPs are known to be associated with risk of various types of cancer, but they have never been investigated in MM. We performed an in silico genome-wide search for miRSNPs predicted to alter binding of miRNAs to their target sequences. We selected 12 miRSNPs and tested their association with MM risk. Our study population consisted of 1,832 controls and 2,894 MM cases recruited from seven European countries and Israel in the context of the IMMEnSE (International Multiple Myeloma rESEarch) consortium. In this population two SNPs showed an association with p

Although having promising anti-myeloma properties, the pan-histone deacetylase inhibitor (HDACi) panobinostat lacks therapeutic activity as a single agent. The aim of the current study was to elucidate the mechanisms underlying multiple myeloma (MM) resistance to panobinostat monotherapy and to define strategies to overcome it. Sensitivity of MM cell lines and primary CD138+ cells from MM patients to panobinostat correlated with reduced expression of the chemokine receptor CXCR4, whereas overexpression of CXCR4 in MM cell lines increased their resistance to panobinostat. Decreased sensitivity to HDACi was associated with reversible G0/G1 cell growth arrest while response was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators revealed the pro-survival mTOR pathway to be regulated by CXCR4 overexpression. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance to HDACi and induced synergistic cell death. The combination of panobinostat/everolimus resulted in sustained DNA damage and irreversible suppression of proliferation accompanied by robust apoptosis. Gene expression analysis revealed distinct genetic profiles of single versus combined agent exposure. Whereas panobinostat increased the expression of the cell cycle inhibitor p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated the expression of DNA repair genes and mitotic checkpoint regulators. Importantly, the combination of panobinostat with everolimus effectively targeted CXCR4-expressing resistant MM cells in vivo in the BM niche. In summary, our results uncover the mechanism responsible for the strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel potential therapeutic approach to treat MM.

Chimeric antigen receptor (CAR) T cells have shown promising results in patients (pts) with B cell malignancies, yet approximately 60% of pts with diffuse large B cell lymphoma (DLBCL) will relapse. Therefore, future efforts are needed to improve the outcomes of these pts. A total of 18 pts with relapsed/refractory B cell malignancies, DLBCL (n=17) and ALL (n=1), were enrolled on a phase 1b/2 study (NCT02772198) of locally produced CD19 CAR T cells. The median age was 40.5 years (range, 23-70). All pts received a lymphodepleting preparative regimen with cyclophosphamide and fludarabine, followed by intravenous infusion of autologous CD19 CAR T cells with a CD28 costimulatory domain. Dosing of CAR T cells was 1-1.5 million CAR+ cells per kg. Clinical response was determined at 28 days following cell administration. Blood samples obtained prior to the lymphodepleting conditioning and at days 7, 14, 21, 30 and 60 after CAR T administration were collected. Cell phenotype was assessed on peripheral blood mononuclear cells (PBMCs) using multiparametric flow cytometry. The manufactured CAR T products (n=9) were also subjected to immunophenotypic analysis. Clinically, 11 of 18 pts (61%) responded to CAR T therapy, 6 (33%) with complete response (CR), and 5 (28%) with partial response (PR). Analysis of manufactured CAR T products (n=9) revealed high CD3+ purity (99%), composed with CD4+ (28%) and CD8+ (72%) T cells. Phenotypic characterization demonstrated marked heterogeneity in the percentages of naïve, central memory, effector memory and effector cells within the CD4+ and CD8+ subsets in the CAR T product. In order to assess the homing potential of CAR T cells, expression of chemokine receptors was evaluated in the product cells. High CXCR3 expression was detected (77% and 96% positive within CD4+ and CD8+ subsets, respectively), indicating high migratory capacity of CAR T cells toward inflamed tissues with high levels of CXCL9 and CXCL10 ligands. Furthermore, co-expression of CXCR4 (56% and 54% positive within CD4+ and CD8+ cells) suggests increased homing ability of the manufactured CAR T toward CXCL12-rich bone marrow microenvironment and lymph nodes. Interestingly, higher CCR7 expression (32% vs 8.5%) and lower CCR6 levels (15% vs 28%) were detected on CD8+ CAR T cells from responding pts who achieved CR and PR (n=6) in comparison to non-responders (n=3), suggesting that less differentiated phenotype together with increased trafficking of CAR T to lymphoid tissue corresponds with improved clinical outcome. Additionally, we assessed the immunoregulatory and senescent/exhausted phenotype in CAR T products. Low percentage of CD4+CD25+CD127- Treg cells (13.5%) was detected, with no correlation to clinical response. However, significantly higher frequency of exhausted CD57+CD39+CD28- cytotoxic CD8+ cells stand out as signature population in CAR T products of non-responders in comparison to CR pts (37% vs 9.5%, p It is known that immunosuppressive environment affects CAR T cell activation, dampening anti-tumor responses. Therefore, we next evaluated the frequency and kinetics of regulatory T cells and myeloid suppressor cells in the peripheral blood of the CAR T treated pts. Notably, responding and non-responding pts presented distinct Treg patterns. Pts achieving CR demonstrated modest and delayed increase in Treg cells, reaching maximal frequency of 23% Treg out of CD4+ cells at day 21 post CAR T infusion, declining to basal low levels (12.5%) at day 30. In contrast, non-responders possessed rapidly increasing percentage of Treg cells (35%) at day 14 post-infusion. In line with this finding, notable increase in proportion of immunosuppressive CD11b+CD14+ myeloid cells expressing CD163, CD206 and MERTK M2 markers was detected in blood of non-responders, while pts achieving CR experienced transient increase in myeloid suppressor cells at day 7 that went back to normal levels at day 14. Overall, these results elucidate in part the mechanisms of CAR T traffic, immunosuppressive responses as well as induction of T cell senescence/exhaustion that most probably downregulate CAR T effectiveness as observed in non-responding pts. It is conceivable that deeper understanding of these processes will help not just establishing surrogate markers predicting clinical responses but may lead to new strategies for restoration of CAR T activation, thereby improving their efficacy and patient's prognosis. Disclosures No relevant conflicts of interest to declare.

Post-transplant cyclophosphamide (PTCy) effectively prevents alloreactive responses from unmanipulated grafts, but its effect on subsequent immune reconstitution remains largely undetermined. A total of 32 patients (pts) (ages > 18) who underwent allogeneic hematopoietic stem cell transplantation (HCT) mainly for acute leukemia and myelodysplastic syndrome were evaluated. Blood samples were collected at 15, 30, 60, 90, 180 and 270 days post-HCT. Cell phenotype was assessed on peripheral blood mononuclear cells (PBMC) using multiparametric flow cytometry. The proliferative and activation T cell capacity in response to mitogenic stimulation was determined. Pts who received graft versus host disease (GVHD) prophylaxis with PTCy/ATG combination (n=8) were compared with pts who received ATG (Grafalon®, Neovii) alone (n=13), and pts undergoing HCT with no ATG (n=11). Five healthy controls served for the functional assays. First, CD4+ and CD8+ T cell differentiation was evaluated assessing CD62L, CD45RA, and CCR7 expression. A smaller percentage of effector cells was detected in the PTCy/ATG group at days 30, 60 and 90 after HCT, compared to the no-ATG and ATG cohorts. Accordingly, the percentage of central memory and effector memory cells was significantly increased in PTCy/ATG pts at the same time points. Next, immuno-modulatory cell populations were assessed. A transient increase in the percentage of CD4+CD25+CD127- Treg cells was detected in both ATG and PTCy/ATG cohorts in the early post-HCT period, reaching maximal frequency of 21% Treg out of the CD4+ subset (range 14-42%) in the ATG group and 19% (range 12-30%) in the PTCy/ATG group on day 21, suggesting that Treg cells are relatively spared by Cy. However, a distinct expression pattern of checkpoint molecules was revealed in pts receiving PTCy/ATG. The percentage of PD1-positive CD4+ and CD8+ cells was comparable in the no-ATG and ATG cohorts (44% vs 45% and 35% vs 41%, respectively). Yet, PD1 expression was significantly increased on both CD4+ (61%, p Furthermore, a significant increase in the percentage of CD28-CD127- cells out of CD8+ cells was detected in PTCy/ATG pts compared to the ATG and no-ATG groups on day 60 post-HCT (82% vs 56% vs 38%, respectively, p Exhausted/senescent T cells showed functional impairment, which manifested as reduced potential for cytokine production and poor proliferative capacity. Notably, both CD4+ and CD8+ cells from ATG and PTCy/ATG cohorts demonstrated diminished proliferation in response to ex-vivo αCD3/αCD28 stimulation, when compared to the no-ATG cohort and healthy controls (p Finally, PTCy/ATG-treated patients demonstrated altered phenotypic and functional recovery of the CD8+ cells. While CD4+ cells gradually downregulated PD1 expression and up-regulated co-stimulatory molecules CD28 and CD127 on days 180 and 270, notably CD8+ cells retained exhausted/tolerogenic phenotype as late as 270 days post-HCT. Our results demonstrate that the acquisition of senescent/exhausted phenotype by cytotoxic CD8+ cells is a distinctive feature of HCT with ATG conditioning. PTCy further compromises recovery of CD8+ cells and impairs NK activation, resulting in a sustained immunosuppression. Hopefully, these results will not only lead to a better understanding of the tolerance mechanisms behind PTCy/ATG anti GVHD prophylaxis but will also assist in the development of novel clinical treatments. Disclosures No relevant conflicts of interest to declare.

// Katia Beider 1 , Hanna Bitner 1 , Merav Leiba 1 , Odit Gutwein 1 , Maya Koren-Michowitz 1 , Olga Ostrovsky 1 , Michal Abraham 3 , Hanna Wald 3 , Eithan Galun 2 , Amnon Peled 2 and Arnon Nagler 1 1 Hematology Division and CBB, Guy Weinshtock Multiple Myeloma Foundation, Chaim Sheba Medical Center, Tel-Hashomer, Israel 2 Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel 3 Biokine Therapeutics Ltd., Science Park, Ness Ziona, Israel Correspondence: Arnon Nagler, email: // Keywords : MM, M2 macrophages, CXCR4 Received : May 21, 2014 Accepted : July 11, 2014 Published : July 12, 2014 Abstract Multiple myeloma (MM) cells specifically attract peripheral-blood monocytes, while interaction of MM with bone marrow stromal cells ( BMSCs) significantly increased monocyte recruitment (p