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Long noncoding RNA differentiation antagonizing nonprotein coding RNA (lncRNA-DANCR) is associated with poor prognosis in multiple cancers, and promotes cancer stemness and invasion. However, the exact mechanisms by which DANCR promotes non-small cell lung cancer (NSCLC) remain elusive. In this study, we determined that DANCR knockdown (KD) impeded cell migration and reduced stem-like characteristics in two NSCLC cell lines, A549 and H1755. Wnt signaling was shown to promote NSCLC proliferation, stemness, and invasion, therefore, we hypothesized that DANCR may regulate these activities through induction of the Wnt/&beta, catenin pathway. DANCR KD reduced &beta, catenin signaling and protein expression, and decreased the expression of &beta, catenin gene targets c-Myc and Axin2. One of the well-defined functions of lncRNAs is their ability to bind and inhibit microRNAs. Through in silico analysis, we identified tumor suppressor miR-216a as a potential binding partner to DANCR, and confirmed this binding through coimmunoprecipitation and luciferase-reporter assays. Furthermore, we show that DANCR-induced &beta, catenin protein expression may be blocked with miR-216a overexpression. Our findings illustrate a role of DANCR in NSCLC migration and stemness, and suggest a novel DANCR/miR-216a signaling axis in the Wnt/&beta, catenin pathway.

Hyperhomocysteinemia (HHcy) is an established and potent independent risk factor for degenerative diseases, including cardiovascular disease (CVD), Alzheimer disease, type II diabetes mellitus, and chronic kidney disease. HHcy has been shown to inhibit proliferation and promote inflammatory responses in endothelial cells (EC), and impair endothelial function, a hallmark for vascular injury. However, metabolic processes and molecular mechanisms mediating HHcy-induced endothelial injury remains to be elucidated. This study examined the effects of HHcy on the expression of microRNA (miRNA) and mRNA in human aortic EC treated with a pathophysiologically relevant concentration of homocysteine (Hcy 500 μM). We performed a set of extensive bioinformatics analyses to identify HHcy-altered metabolic and molecular processes. The global functional implications and molecular network were determined by Gene Set Enrichment Analysis (GSEA) followed by Cytoscape analysis. We identified 244 significantly differentially expressed (SDE) mRNA, their relevant functional pathways, and 45 SDE miRNA. HHcy-altered SDE inversely correlated miRNA-mRNA pairs (45 induced/14 reduced mRNA) were discovered and applied to network construction using an experimentally verified database. We established a hypothetical model to describe the biochemical and molecular network with these specified miRNA/mRNA axes, finding: 1) HHcy causes metabolic reprogramming by increasing glucose uptake and oxidation, by glycogen debranching and NAD+/CoA synthesis, and by stimulating mitochondrial reactive oxygen species production via NNT/IDH2 suppression-induced NAD+/NADP-NADPH/NADP+ metabolism disruption; 2) HHcy activates inflammatory responses by activating inflammasome-pyroptosis mainly through ↓miR193b→↑CASP-9 signaling and by inducing IL-1β and adhesion molecules through the ↓miR29c→↑NEDD9 and the ↓miR1256→↑ICAM-1 axes, as well as GPCR and interferon α/β signaling; 3) HHcy promotes cell degradation by the activation of lysosome autophagy and ubiquitin proteasome systems; 4) HHcy causes cell cycle arrest at G1/S and S/G2 transitions, suppresses spindle checkpoint complex and cytokinetic abscission, and suppresses proliferation through ↓miRNA335/↑VASH1 and other axes. These findings are in accordance with our previous studies and add a wealth of heretofore-unexplored molecular and metabolic mechanisms underlying HHcy-induced endothelial injury. This is the first study to consider the effects of HHcy on both global mRNA and miRNA expression changes for mechanism identification. Molecular axes and biochemical processes identified in this study are useful not only for the understanding of mechanisms underlying HHcy-induced endothelial injury, but also for discovering therapeutic targets for CVD in general., Graphical abstract Image 1, Highlights • Identified multiple HHcy-altered metabolic and molecular processes potentially responsible for HHcy-induced endothelial injury via examining global mRNA/miRNA expression changes in Hcy-treated EC and performing comprehensive bioinformatic studies. • HHcy may activate glucose uptake signaling via the ↓miR148b→↑SLC2A axis. • HHcy may induce glucose oxidation signaling by switching pyruvate metabolism from lactate synthesis to mitochondrial oxidation via expression changes of ↑MPC1 & ↓LDHB. • HHcy may disrupt redox homeostasis mostly by suppressing NNT/IDH2-related mt-NADPH/mt-NAD+ signaling. • HHcy may increase FA β-oxidation, glutamine, TCA cycle and OXPHOS signaling. • HHcy may activate inflammatory signaling via the ↓miR29c→↑NEDD9 and the ↓miR1256→↑ICAM-1 axes. • HHcy may activate inflammasome/pyroptosis-related signaling by the ↓miR137→↑TLR3, the ↓miR574→↑TRAF5, and the ↓miR193b→↑CASP-9 axes, and induce IL1α/β and CASP-10/7. • HHcy may induce inflammation signaling via GPCR activation through the ↓miRNA335→↑CXCR4/↑GNA14 axes. • HHcy may activate molecular degradation process signaling through the ↓miRNA335→↑ASAH1/↑ABCB9 axes. • HHcy may suppress cell cycle and proliferation through the miR491→↓HMGA2→↓CCNA2/CCNB2, the ↓miR335→↑VASH1, the ↓miR181a→↑PHLDA1, the miR6045→↓CENPH, the miR22→↓PRR11/↓BRCA2, and the miR605/miR497/miR514a→CEP55 axes

The use of imatinib, second and third generation ABL tyrosine kinase inhibitors (TKI) (i.e. dasatinib, nilotinib, bosutinib and ponatinib) made CML a clinically manageable and, in a small percentage of cases, a cured disease. TKI therapy also turned CML blastic transformation into a rare event; however, disease progression still occurs in those patients who are refractory, not compliant with TKI therapy or develop resistance to multiple TKIs. In the past few years, it became clear that the BCR-ABL1 oncogene does not operate alone to drive disease emergence, maintenance and progression. Indeed, it seems that bone marrow (BM) microenvironment-generated signals and cell autonomous BCR-ABL1 kinase-independent genetic and epigenetic alterations all contribute to: i. persistence of a quiescent leukemic stem cell (LSC) reservoir, ii. innate or acquired resistance to TKIs, and iii. progression into the fatal blast crisis stage. Herein, we review the intricate leukemic network in which aberrant, but finely tuned, survival, mitogenic and self-renewal signals are generated by leukemic progenitors, stromal cells, immune cells and metabolic microenvironmental conditions (e.g. hypoxia) to promote LSC maintenance and blastic transformation.

The human immunodeficiency virus (HIV) is a lentivirus that infects cells of the immune system, resulting in cell death and loss of cell-mediated immunity. When the number of immune cells drops below a critical population, HIV can result in the acquired immunodeficiency syndrome (AIDS), wherein opportunistic infections and cancers flourish in the absence of immune surveillance. Worldwide, there are over 30 million people infected with HIV, resulting in a significant global health burden (1). The current standard of care for HIV is combined antiretroviral therapy (cART), and while this effectively prevents disease progression and death, treatment must be continued for the rest of the patient’s life (2). Therefore, new therapies that can eradicate and prevent the disease are a crucial area of research in global health.

Endothelial lipase (LIPG) plays a critical role in lipoprotein metabolism, cytokine expression, and the lipid composition of cells. Thus far, the extensive investigations of LIPG have focused on its mechanisms and involvement in metabolic syndromes such as atherosclerosis. However, recent developments have found that LIPG plays a role in cancer. This review summarizes the field of LIPG study. We focus on the role of LIPG in lipid metabolism and the inflammatory response, and highlight the recent insights in its involvement in tumor progression. Finally, we discuss potential therapeutic strategies for targeting LIPG in cancer, and the therapeutic potential of LIPG as a drug target.

The normal myoepithelium has a tumor-suppressing nature and inhibits the progression of ductal carcinoma in situ (DCIS) into invasive ductal carcinoma (IDC). Conversely, a growing number of studies have shown that tumor-associated myoepithelial cells have a tumor-promoting effect. Moreover, the exact role of tumor-associated myoepithelial cells in the DCIS-to-IDC development remains undefined. To address this, we explored the role of tumor-associated myoepithelial cells in the DCIS-to-IDC progression. We developed a direct coculture system to study the cell-cell interactions between DCIS cells and tumor-associated myoepithelial cells. Coculture studies indicated that tumor-associated myoepithelial cells promoted the invasive progression of a DCIS cell model in vitro, and mechanistic studies revealed that the interaction with DCIS cells stimulated tumor-associated myoepithelial cells to secrete TGFβ1, which subsequently contributed to activating the TGFβ/Smads pathway in DCIS cells. We noted that activation of the TGFβ signaling pathway promoted the epithelial-mesenchymal transition, basal-like phenotypes, stemness, and invasiveness of DCIS cells. Importantly, xenograft studies further demonstrated that tumor-associated myoepithelial cells enhanced the DCIS-to-IDC progression in vivo. Furthermore, we found that TGFβ-mediated induction of oncogenic miR-10b-5p expression and down-regulation of RB1CC1, a miR-10b-5p-targeted tumor-suppressor gene, contributed to the invasive progression of DCIS. Our findings provide the first experimental evidence to directly support the paradigm that altered DCIS-associated myoepithelial cells promote the invasive progression of DCIS into IDC via TGFβ signaling activation.

BackgroundInjury due to myocardial infarction (MI) is largely irreversible. Once an infarct has occurred, the clinical goal becomes limiting remodeling, preserving left ventricular function, and preventing heart failure. Although traditional approaches (e.g., β-blockers) partially preserve left ventricular function, novel strategies are needed to limit ventricular remodeling post-MI.ObjectivesThe aim of this study was to determine the role of glycogen synthase kinase–3α (GSK-3α) in post-MI remodeling.MethodsMice with cardiomyocyte-specific conditional deletion of Gsk3α and littermate controls underwent sham or MI surgery. Heart function was assessed using serial M-mode echocardiography.ResultsGsk3α deletion in the heart markedly limits remodeling and preserves left ventricular function post-MI. This is due at least in part to dramatic thinning and expansion of the scar in the control hearts, which was less in the heart of knockout (KO) mice. In contrast, the border zone in the KO mice demonstrated a much thicker scar, and there were more viable cardiomyocytes within the scar/border zone. This was associated with less apoptosis and more proliferation of cardiomyocytes in the KO mice. Mechanistically, reduced apoptosis was due, at least in part, to a marked decrease in the Bax/Bcl-2 ratio, and increased cardiomyocyte proliferation was mediated through cyclin E1 and E2F-1 in the hearts of the KO mice.ConclusionsTaken together, these findings show that reducing GSK-3α expression in cardiomyocytes limits ventricular remodeling and preserves cardiac function post-MI. Specifically targeting GSK-3α could be a novel strategy to limit adverse remodeling and heart failure.

Background— Myocardial infarction–induced remodeling includes chamber dilatation, contractile dysfunction, and fibrosis. Of these, fibrosis is the least understood. After myocardial infarction, activated cardiac fibroblasts deposit extracellular matrix. Current therapies to prevent fibrosis are inadequate, and new molecular targets are needed. Methods and Results— Herein we report that glycogen synthase kinase-3β (GSK-3β) is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using 2 fibroblast-specific GSK-3β knockout mouse models, we show that deletion of GSK-3β in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Deletion of GSK-3β induces a profibrotic myofibroblast phenotype in isolated cardiac fibroblasts, in post–myocardial infarction hearts, and in mouse embryonic fibroblasts deleted for GSK-3β. Mechanistically, GSK-3β inhibits profibrotic transforming growth factor-β1/SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3β resulted in the significant increase of SMAD-3 transcriptional activity. This pathway is central to the pathology because a small-molecule inhibitor of SMAD-3 largely prevented fibrosis and limited left ventricular remodeling. Conclusions— These studies support targeting GSK-3β in myocardial fibrotic disorders and establish critical roles of cardiac fibroblasts in remodeling and ventricular dysfunction.

β-Adrenergic receptor (βAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to relay pro-survival effects via unknown mechanisms. We hypothesized that acute βAR-mediated EGFR transactivation in the heart promotes differential subcellular activation of ERK1/2 and Akt, promoting cell survival through modulation of apoptosis. C57BL/6 mice underwent acute i.p. injection with isoproterenol (ISO)±AG 1478 (EGFR antagonist) to assess the impact of βAR-mediated EGFR transactivation on the phosphorylation of ERK1/2 (P-ERK1/2) and Akt (P-Akt) in distinct cardiac subcellular fractions. Increased P-ERK1/2 and P-Akt were observed in cytosolic, plasma membrane and nuclear fractions following ISO stimulation. Whereas the P-ERK1/2 response was EGFR-sensitive in all fractions, the P-Akt response was EGFR-sensitive only in the plasma membrane and nucleus, results confirmed in primary rat neonatal cardiomyocytes (RNCM). βAR-mediated EGFR-transactivation also decreased apoptosis in serum-depleted RNCM, as measured via TUNEL as well as caspase 3 activity/cleavage, which were sensitive to the inhibition of either ERK1/2 (PD184352) or Akt (LY-294002) signaling. Caspase 3 activity/cleavage was also sensitive to the inhibition of transcription, which, with an increase in nuclear P-ERK1/2 and P-Akt in response to ISO, suggested that βAR-mediated EGFR transactivation may regulate apoptotic gene transcription. An Apoptosis PCR Array identified tnfsf10 (TRAIL) to be altered by ISO in an EGFR-sensitive manner, results confirmed via RT-PCR and ELISA measurement of both membrane-bound and soluble cardiomyocyte TRAIL levels. βAR-mediated EGFR transactivation induces differential subcellular activation of ERK1/2 and Akt leading to increased cell survival through the modulation of caspase 3 activity and apoptotic gene expression in cardiomyocytes.

β-adrenergic receptor (βAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to promote cardioprotection in a mouse model of heart failure and we recently showed that this mechanism leads to enhanced cell survival in part via regulation of apoptotic transcript expression in isolated primary rat neonatal cardiomyocytes. Thus, we hypothesized that this process could regulate cardiac transcript expression in vivo. To comprehensively assess cardiac transcript alterations in response to acute βAR-dependent EGFR transactivation, we performed whole transcriptome analysis of hearts from C57BL/6 mice given i.p. injections of the βAR agonist isoproterenol in the presence or absence of the EGFR antagonist gefitinib for 1 hour. Total cardiac RNA from each treatment group underwent transcriptome analysis, revealing a substantial number of transcripts regulated by each treatment. Gefitinib alone significantly altered the expression of 405 transcripts, while isoproterenol either alone or in conjunction with gefitinib significantly altered 493 and 698 distinct transcripts, respectively. Further statistical analysis was performed, confirming 473 transcripts whose regulation by isoproterenol were significantly altered by gefitinib (isoproterenol-induced up/downregulation antagonized/promoted by gefinitib), including several known to be involved in the regulation of numerous processes including cell death and survival. Thus, βAR-dependent regulation of cardiac transcript expression in vivo can be modulated by the EGFR antagonist gefitinib.

Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. β-adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous βAR in primary neonatal rat cardiac fibroblasts (NRCF). The EPIC-BT by Corning was used to measure DMR responses in primary isolated NRCF treated with various βAR and EGFR ligands. Additional molecular assays for cAMP generation and receptor internalization responses were used to correlate the DMR findings with established βAR signaling pathways. Catecholamine stimulation of NRCF induced a concentration-dependent negative DMR deflection that was competitively blocked by βAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Subtype-selective βAR ligand profiling revealed a dominant role for β2AR in mediating the DMR responses, consistent with the relative expression levels of β2AR and β1AR in NRCF. βAR-mediated cAMP generation profiles revealed similar kinetics to DMR responses, each of which were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, G protein-independent βAR signaling through epidermal growth factor receptor (EGFR) was assessed, revealing a smaller but significant contribution of this pathway to the DMR response to βAR stimulation. Measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous βAR signaling via both G protein-dependent and –independent pathways.

β-adrenergic receptors (βAR) are critical regulators of cardiac function whose dysregulation during heart failure are associated with diminished function. However, βAR-mediated EGFR transactivation has been shown to relay cardioprotection in a mouse model of heart failure via unknown mechanisms. We hypothesized that transactivation of EGFR promotes survival via distinct cardiomyocyte signaling responses leading to decreases in apoptosis. To test this hypothesis, C57BL/6 mice were injected with isoproterenol (Iso) in the presence or absence of the EGFR antagonist AG1478 and ERK1/2 and Akt phosphorylation and subcellular distribution were assessed. Following 10 min Iso stimulation, increases in ERK1/2 and Akt phosphorylation were observed in cytosolic, plasma membrane and nuclear fractions. Phosphorylation of ERK1/2 were AG1478 sensitive in all three fractions while Akt phosphorylation occurred through EGFR-transactivation only in plasma membrane and nuclear fractions, which was confirmed in rat neonatal cardiomyocytes (RNCM). Additionally, EGFR-transactivation by βAR decreased apoptosis, as measured via caspase 3 activation/activity and TUNEL assay, which was sensitive to inhibition of both ERK1/2 and Akt signaling pathways. Increased phosphorylation of ERK1/2 and Akt in the nucleus and the ability to inhibit Iso-mediated changes in apoptosis with the transcriptional inhibitor Actinomycin D suggested that the cardioprotective effects of Iso-mediated EGFR transactivation may be influenced by changes in gene transcription. An Apoptotsis RT2 PCR Array was used to identify changes in transcript levels of 84 apoptotic genes. Of these, 12 were found to be altered in response to EGFR inhibition in the presence of Iso. These results demonstrate that βAR-mediated EGFR transactivation in the heart induces differential subcellular activation of ERK1/2 and Akt and leads to the promotion of cell survival, in part through the modulation of apoptotic gene expression in cardiomyocytes. Further understanding the downstream consequences of these effects in response to βAR-mediated EGFR transactivation could lead to improved therapies for the treatment of heart failure.

The L-α-lysophosphatidylinositol (LPI)-sensitive receptor GPR55 is coupled to Ca2+ signaling. Low levels of GPR55 expression in the heart have been reported. Similar to other G protein-coupled receptors involved in cardiac function, GPR55 may be expressed both at the sarcolemma and intracellularly. Thus, to explore the role of GPR55 in cardiomyocytes, we used calcium and voltage imaging and extracellular administration or intracellular microinjection of GPR55 ligands. We provide the first evidence that, in cultured neonatal ventricular myocytes, LPI triggers distinct signaling pathways via GPR55, depending on receptor localization. GPR55 activation at the sarcolemma elicits, on one hand, Ca2+ entry via L-type Ca2+ channels and, on the other, inositol 1,4,5-trisphosphate-dependent Ca2+ release. The latter signal is further amplified by Ca2+-induced Ca2+ release via ryanodine receptors. Conversely, activation of GPR55 at the membrane of intracellular organelles promotes Ca2+ release from acidic-like Ca2+ stores via the endolysosomal NAADP-sensitive two-pore channels. This response is similarly enhanced by Ca2+-induced Ca2+ release via ryanodine receptors. Extracellularly applied LPI produces Ca2+-independent membrane depolarization, whereas the Ca2+ signal induced by intracellular microinjection of LPI converges to hyperpolarization of the sarcolemma. Collectively, our findings point to GPR55 as a novel G protein-coupled receptor regulating cardiac function at two cellular sites. This work may serve as a platform for future studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders. Background: The LPI-sensitive receptor GPR55 signals through Ca2+. Results: Activation of sarcolemmal versus intracellular GPR55 mobilizes Ca2+ from distinct pools and associates with cardiomyocyte depolarization and hyperpolarization, respectively. Conclusion: GPR55 location critically affects LPI-induced modulation of cardiomyocyte function. Significance: We identify GPR55 as a new receptor regulating cardiac function at two cellular sites.

Tyrosine kinase inhibitors (TKIs) combined with chemotherapy significantly improved outcomes in adult Philadelphia-chromosome-positive (Ph+) B-cell Acute Lymphoblastic Leukemia (B-ALL). However, high relapse rates due to development of TKI resistance or chemotherapy-induced adverse effects remain the major therapeutic challenges. Furthermore, all TKIs are not effective against Ph+leukemia-initiating cells (LICs). The tumor suppressor protein phosphatase 2A (PP2A) is inactive in almost all solid and hematopoietic tumors. Suppression of PP2A activity correlates with poor outcome and disease progression, and largely relies on the aberrant expression of CIP2A, SET and/or SETBP1. SETBP1 was discovered as a SET-interacting protein, and recently described as mutated or overexpressed in several myeloid malignancies where it acts as an independent negative prognostic factor and as an inhibitor of PP2A, however its role in lymphoid malignancies is still unknown. Thus, we postulated that SETBP1 regulates survival and self-renewal of Ph+B-ALL LICs and progenitors through inhibition of PP2A. We reported that BCR-ABL1 kinase-dependent and -independent mechanisms induce SET-dependent PP2A inhibition in Ph+ (CMLand B-ALL) progenitors and quiescent TKI-resistant CML LICs, respectively, and that SET downregulation or pharmacologic (i.e. SET-interacting PP2A-activating drugs; PADs) restoration of PP2A activity strongly impaired malignant but not normal hematopoiesis by selectively killing Ph+progenitors (CML and ALL) and TKI-resistant quiescent stem (CML) cells. Here, we show that wild type SETBP1 is markedly induced in an imatinib (IM)-insensitive manner in primary CD34+CD19+ Ph+ B-ALL progenitors and Ph+ B-ALL cell lines (BV173 and SUP-B15) and barely detectable in CD34+ cells from CML patients in chronic and blastic phase. Overexpression of SETBP1 was found essential for PP2A inhibition in Ph+ B-ALL blasts. Accordingly, shRNA-dependent SETBP1 downregulation impaired clonogenic potential and self-renewal of CD34+CD19+ Ph+ B-ALL cells in CFC and serial replating assays. Furthermore, we have evidence that a SETBP1-SET/CIP2A inhibitory complex may exist in Ph+ cells, suggesting that SETBP1 might serve to recruit SET and CIP2A to suppress PP2A activity. Indeed, we found that CIP2A, like SET and SETBP1, is also overexpressed in CD34+CD19+ Ph+ B-ALL compared to CD34+CD19+cell from BM of healthy donors. Because, SETBP1 stabilizes SET and augments PP2A inhibition, and ectopic SETBP1 expression confers self-renewal to mouse myeloid progenitors and cooperates with BCR-ABL1 to induce a CML blast crisis-like disease in mice, our data suggest that aberrant SETBP1 expression might significantly contributes to the development of Ph+ B-ALL and persistence of TKI-resistant Ph+ B-ALL LICs. Disclosures Milojkovic: Ariad: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Roy:Paladin: Consultancy; Fate Therapeutics: Consultancy; Novartis: Consultancy; Kiadis Pharma: Research Funding.

MiR-300 is a microRNA predicted to target multiple components of the BCR-ABL1/JAK2/hnRNPA1/SET/PP2A/β-catenin pathway, which is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs). Nanostring arrays analysis of bone marrow (BM) cells from healthy individuals (n = 5) and CML patients (n = 10) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300). MiR-300 transduction in CMLCD34+ cells and BCR-ABL1+ cell lines decreased JAK2, β-catenin, hnRNPA1 and SET expression and increased PP2A activity. Targets were confirmed by miR-300 expression in BCR-ABL1+ cells expressing Flag-tagged miR-300-targets lacking or carrying a wild-type or mutated 3’UTR. Restored miR-300 expression in CMLCD34+ cells and/or BCR-ABL1+ cell lines impaired proliferation and clonogenic potential, markedly reduced LTC-ICs, and increased TKI sensitivity. Notably, miR-300 expression was inhibited by BCR-ABL1 in proliferating cells. Accordingly, imatinib restored miR-300 expression in CD34+ dividing progenitors and BCR-ABL1+ cell lines without altering miR-300 levels in quiescent (CFSEMAX) CMLCD34+ cells (n = 3), consistent with the BCR-ABL1 kinase-dependent activation of the Jak2/SET/PP2A/β-catenin pathway in CML progenitors but not quiescent Ph+ HSCs. Surprisingly, miR-300 levels were increased in CD34+CD38- compared to CD34+CD38+ CML cells, and >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n = 4). To determine whether enhanced miR-300 expression in quiescent cells depends on cell autonomous events or is induced by the BM microenvironment, we exposed BCR-ABL+ cells to conditioned medium (CM) of HS-5 or hTERT mesenchymal stem cells (MSC). CM strongly decreased proliferation, induced imatinib but not FTY720 (PP2A activator) resistance, increased miR-300 levels, decreased BCR-ABL1 activity and Jak2 expression but not its activity, and did not alter β-catenin levels or PP2A activity. Interestingly, miR-300 was found in MSC-derived exosomes, and its expression increased in BCR-ABL1+ cells exposed to exosomes. Accordingly, proliferation of CML-BCCD34+and LAMA-84 cells was strongly reduced upon exposure to MSC-derived exosomes. These effects were abolished when we used CM from MSCs transduced with a miR-300 antagomir. Altogether our results indicate that downregulation of miR-300 appears necessary for the activation of JAK2/SET/PP2A/β-catenin survival signals in CML progenitors. Conversely, increased miR-300 levels (endogenous and MSC-derived) seem to be required for HSC quiescence. Citation Format: Rossana Trotta, Giovannino Silvestri, Lorenzo Stramucci, Justin J. Ellis, Justine Yu, Jason J. Harb, Paolo Neviani, Guido Marcucci, Klara Srutova, Polakova K. Machova, Denis-Claude Roy, Peter Hokland, Michael Deininger, Ravi Bhatia, Carlo Gambacorti-Passerini, Dragana Milojkovic, Alistair Reid, Jane Apperley, Ferenc Livak, Jianfei Qi, Maria R. Baer, Danilo Perrotti. Role of the MSC-derived exosomal and endogenous JAK2-SET/PP2A-β-catenin-modulator miR-300 in leukemic stem/progenitor proliferation and survival in CML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 951.

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.

MiR-300 is a microRNA predicted to target multiple components of the BCR-ABL1 / JAK2 / hnRNPA1 / SET / PP2A / β-catenin pathway, which is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs). Nanostring arrays analysis of bone marrow (BM) cells from healthy individuals (n=5) and CML patients (n=10) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300). MiR-300 transduction in CMLCD34+ cells and BCR-ABL1+ cell lines decreased JAK2, β-catenin, hnRNPA1 and SET expression and increased PP2A activity. Targets were confirmed by miR-300 expression in BCR-ABL1+ cells expressing Flag-tagged miR-300-targets lacking or carrying a wild-type or mutated 3'UTR. Restored miR-300 expression in CMLCD34+ cells and/or BCR-ABL1+ cell lines impaired cell cycle progression, proliferation and clonogenic potential, markedly reduced LTC-ICs, and increased TKI sensitivity. Notably, miR-300 expression was inhibited by BCR-ABL1 in proliferating cells. Accordingly, imatinib restored miR-300 expression in CD34+ dividing progenitors and BCR-ABL1+ cell lines without altering miR-300 levels in quiescent (CFSEMAX) CMLCD34+ cells (n=3), consistent with the BCR-ABL1 kinase-dependent activation of the Jak2/SET/PP2A/β-catenin pathway in CML progenitors but not quiescent Ph+ HSCs. Indeed, ectopic SET expression counteracted the negative effects of mir-300 on cell proliferation and survival. Surprisingly, miR-300 levels were increased in CD34+CD38- compared to CD34+CD38+ CML cells, and >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n=4). To determine whether enhanced miR-300 expression in quiescent cells depends on cell autonomous events or is induced by the BM microenvironment, we exposed BCR-ABL+ cells to conditioned medium (CM) of HS-5 or hTERT mesenchymal stem cells (MSC). CM strongly decreased proliferation, induced imatinib but not FTY720 (PP2A activator) resistance, increased miR-300 levels, decreased BCR-ABL1 activity and Jak2 expression but not its activity, and did not alter b-catenin levels or PP2A activity. Interestingly, miR-300 was found in MSC-derived exosomes, and its expression increased in BCR-ABL1+ cells exposed to exosomes. Accordingly, proliferation of CML-BCCD34+ and LAMA-84 cells was strongly reduced upon exposure to MSC-derived exosomes. These effects were abolished when we used CM from MSCs transduced with a miR-300 antagomir. Altogether our results indicate that downregulation of miR-300 appears necessary for the activation of JAK2/SET/PP2A/b-catenin survival signals in CML progenitors. Conversely, increased miR-300 levels (endogenous and MSC-derived) seem to be required for HSC quiescence. Disclosures Deininger: Novartis: Other: Consulting or Advisory Role, Research Funding; BMS: Other: Consulting & Advisory Role, Research Funding; Celgene: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; ARIAD Pharmaceutical Inc.: Other: Consulting or Advisory Role; Incyte: Other: Consulting or Advisory Role; Pfizer: Other: Consulting or Advisory Role. Milojkovic:BMS: Honoraria; ARIAD Pharmaceuticals Inc.: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.