UM171 suppresses breast cancer progression by inducing KLF2.

PURPOSE: Breast cancer is the most frequent cancer in women with significant death rate. Morbidity is associated with drug resistance and metastasis. Development of novel drugs is unmet need. The aim of this study is to show potent anti-neoplastic activity of the UM171 compound on breast cancer cells and its mechanism of action. METHODS: The inhibitory effect of UM171 on several breast cancer (BC) cell lines was examined using MTT and colony-forming assays. Cell cycle and apoptosis assays were utilized to determine the effect of UM171 on BC cell proliferation and survival. Wound healing scratch and transwell migration assays were used to examine the migration of BC cell lines in culture. Xenograft of mouse model with 4T1 cells was used to determine inhibitory effect of UM171 in vivo. Q-RT-PCR and western blotting were used to determine the expression level of genes effected by UM171. Lentivirus-mediated shRNAs were used to knockdown the expression of KLF2 in BC cells. RESULTS: UM171 was previously identified as a potent agonist of human hematopoietic stem cell renewal and inhibitor of leukemia. In this study, UM171 was shown to inhibit the growth of multiple breast cancer cell lines in culture. UM171-mediated growth inhibition was associated with the induction of apoptosis, G2/M cell cycle arrest, lower colony-forming capacity, and reduced motility. In a xenotransplantation model of mouse triple-negative breast cancer 4T1 cells injected into syngeneic BALB/c mice, UM171 strongly inhibited tumor growth at a level comparable to control paclitaxel. UM171 increased the expression of the three PIM genes (PIM1-3) in breast cancer cells. Moreover, UM171 strongly induced the expression of the tumor suppressor gene KLF2 and cell cycle inhibitor P21CIP1. Accordingly, knockdown of KLF2 using lentivirus-mediated shRNA significantly attenuated the growth suppressor activity of UM171. As PIM1-3 act as oncogenes and are involved in breast cancer progression, induction of these kinases likely impedes the inhibitory effect of KLF2 induction by UM171. Accordingly, combination of UM171 with a PAN-PIM inhibitor LGH447 significantly reduced tumor growth in culture. CONCLUSION: These results suggested that UM171 inhibited breast cancer progression in part through activation of KLF2 and P21. Combination of UM171 with a PAN-PIM inhibitor offer a novel therapy for aggressive forms of breast cancer.

FLI1 induces erythroleukemia through opposing effects on UBASH3A and UBASH3B expression.

BACKGROUND: FLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1. METHODS: Promoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index. RESULTS: Knockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B. CONCLUSIONS: FLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.

Lovastatin inhibits erythroleukemia progression through KLF2-mediated suppression of MAPK/ERK signaling.

BACKGROUND: Lovastatin, an HMG-CoA inhibitor and an effective cholesterol lowering drug, exhibits anti-neoplastic activity towards several types of cancer, although the underlying mechanism is still not fully understood. Herein, we investigated mechanism of growth inhibition of leukemic cells by lovastatin. METHODS: RNAseq analysis was used to explore the effect of lovastatin on gene expression in leukemic cells. An animal model of leukemia was used to test the effect of this statin in vivo. FAM83A and DDIT4 expression was knocked-downed in leukemia cells via lentivirus-shRNA. Western blotting, RT-qPCR, cell cycle analysis and apoptosis assays were used to determine the effect of lovastatin-induced growth suppression in leukemic cells in vitro. RESULTS: Lovastatin treatment strongly inhibited cancer progression in a mouse model of erythroleukemia induced by Friend virus. In tissue culture, lovastatin inhibited cell proliferation through induction of G1 phase cell cycle arrest and apoptosis. Interestingly, lovastatin induced most known genes associated with cholesterol biosynthesis in leukemic cells. Moreover, it suppressed ERK1/2 phosphorylation by downregulating FAM83A and DDIT4, two mediators of MAP-Kinase signaling. RNAseq analysis of lovastatin treated leukemic cells revealed a strong induction of the tumor suppressor gene KLF2. Accordingly, lentivirus-mediated knockdown of KLF2 antagonized leukemia cell suppression induced by lovastatin, associated with higher ERK1/2 phosphorylation compared to control. We further show that KLF2 induction by lovastatin is responsible for lower expression of the FAM83A and DDIT4 oncogenes, involved in the activation of ERK1/2. KLF2 activation by lovastatin also activated a subset of cholesterol biosynthesis genes that may further contribute to leukemia suppression. CONCLUSIONS: These results implicate KLF2-mediated FAM83A/DDIT4/MAPK suppression and activation of cholesterol biosynthesis as the mechanism of leukemia cell growth inhibition by lovastatin.

High-performance laser power converts for direct-energy applications.

Six-junction GaAs laser power converts (LPCs) were designed and fabricated. Each subcell is vertically connected by p++-AlGaAs: C/n++-AlGaAs: Si: Te (1:2) tunnel junction with good thermal stability and a record peak tunneling current density of 1867 A/cm2. The I-V characteristics of LPCs with an aperture of 10×10 mm2 were investigated as a function of laser power and temperature. Maximum conversion efficiency and output power of 57.7% and 15.4 W, respectively, and a continuous stable operation at 22.9 W for over 550 hours were demonstrated. The temperature coefficient of conversion efficiency and open-circuit voltage were -0.197%abs/°C and -8.15 mV/°C, respectively, under 808 nm laser illumination of 21.0 W. Furthermore, an array of 100 large-scale (41×46 mm2) LPCs with an output power of 179 W under 1 kW laser irradiation at 20 m wireless transmission was developed.

1064†nm InGaAs metamorphic laser power converts with over 44% efficiency.

InGaAs metamorphic laser power converters (LPCs) have the potential to deliver electrical energy over distances of several kilometers. In this study, metalorganic chemical vapor deposition (MOCVD) was used to grow InGaAs-based LPCs with an absorption wavelength of 1064 nm. At step thicknesses of 2800 nm, overshoot thicknesses of 6000 nm, reverse component and thicknesses of 2.4% and 700 nm, respectively, a surface roughness of 6.0 nm and InGaAs (24%) lattice relaxation of 93.7% of the InGaAs metamorphic buffer were obtained. The I-V characteristics of LPCs with 10 × 10 mm2 apertures were investigated as a function of laser power and temperature. The maximum conversion efficiency of 44.1% and 550 hours of continuous stable operation at 4 W were demonstrated. Under 1064 nm laser illumination of 4 W, the temperature coefficients for the conversion efficiency and open-circuit voltage were -0.1%abs/°C and -1.6 mV/°C, respectively, and the LPC output power fluctuation was less than 0.5% during 216 hours of continuous temperature change from 20 to 100°C.

ERK activation via A1542/3 limonoids attenuates erythroleukemia through transcriptional stimulation of cholesterol biosynthesis genes.

BACKGROUND: Cholesterol plays vital roles in human physiology; abnormal levels have deleterious pathological consequences. In cancer, elevated or reduced expression of cholesterol biosynthesis is associated with good or poor prognosis, but the underlying mechanisms are largely unknown. The limonoid compounds A1542 and A1543 stimulate ERK/MAPK by direct binding, leading to leukemic cell death and suppression of leukemia in mouse models. In this study, we investigated the downstream consequences of these ERK/MAPK agonists in leukemic cells. METHODS: We employed RNAseq analysis combined with Q-RT-PCR, western blot and bioinformatics to identify and confirm genes whose expression was altered by A1542 and A1543 in leukemic cells. ShRNA lentiviruses were used to silence gene expression. Cell culture and an animal model (BALB/c) of erythroleukemia induced by Friend virus were utilized to validate effects of cholesterol on leukemia progression. RESULTS: RNAseq analysis of A1542-treated cells revealed the induction of all 18 genes implicated in cholesterol biosynthesis. Expression of these cholesterol genes was blocked by cedrelone, an ERK inhibitor. The cholesterol inhibitor lovastatin diminished ERK/MAPK activation by A1542, thereby reducing leukemic cell death induced by this ERK1/2 agonist. Growth inhibition by cholesterol was observed both at the intracellular level, and when orally administrated into a leukemic mouse model. Both HDL and LDL also suppressed leukemogenesis, implicating these lipids as important prognostic markers for leukemia progression. Mechanistically, knockdown experiments revealed that the activation of SREBP1/2 by A1542-A1543 was responsible for induction of only a sub-set of cholesterol biosynthesis genes. Induction of other regulatory factors by A1542-A1543 including EGR1, AP1 (FOS + JUN) LDLR, IER2 and others may cooperate with SREBP1/2 to induce cholesterol genes. Indeed, pharmacological inhibition of AP1 significantly inhibited cholesterol gene expression induced by A1542. In addition to leukemia, high expression of cholesterol biosynthesis genes was found to correlate with better prognosis in renal cancer. CONCLUSIONS: This study demonstrates that ERK1/2 agonists suppress leukemia and possibly other types of cancer through transcriptional stimulation of cholesterol biosynthesis genes.

Erythropoietin Signaling in the Microenvironment of Tumors and Healthy Tissues.

Erythropoietin (EPO), the primary cytokine of erythropoiesis, stimulates both proliferation and differentiation of erythroid progenitors and their maturation to red blood cells. Basal EPO levels maintain the optimum levels of circulating red blood cells. However, during hypoxia, EPO secretion and its expression is elevated drastically in renal interstitial fibroblasts, thereby increasing the number of erythroid progenitors and accelerating their differentiation to mature erythrocytes. A tight regulation of this pathway is therefore of paramount importance. The biological response to EPO is commenced through the involvement of its cognate receptor, EPOR. The receptor-ligand complex results in homodimerization and conformational changes, which trigger downstream signaling events and cause activation or inactivation of critical transcription factors that promote erythroid expansion. In recent years, recombinant human EPO (rEPO) has been widely used as a therapeutic tool to treat a number of anemias induced by infection, and chemotherapy for various cancers. However, several studies have uncovered a tumor promoting ability of EPO in man, which likely occurs through EPOR or alternative receptor(s). On the other hand, some studies have demonstrated a strong anticancer activity of EPO, although the mechanism still remains unclear. A thorough investigation of EPOR signaling could yield enhanced understanding of the pathobiology for a variety of disorders, as well as the potential novel therapeutic strategies. In this chapter, in addition to the clinical relevance of EPO/EPOR signaling, we review its anticancer efficacy within various tumor microenvironments.

Selective ERK1/2 agonists isolated from Melia azedarach with potent anti-leukemic activity.

BACKGROUND: MAPK/ERK kinases transmit signals from many growth factors/kinase receptors during normal cell growth/differentiation, and their dysregulation is a hallmark of diverse types of cancers. A plethora of drugs were developed to block this kinase pathway for clinical application. With the exception of a recently identified agent, EQW, most of these inhibitors target upstream factors but not ERK1/2; no activator of ERK1/2 is currently available. METHOD: A library of compounds isolated from medicinal plants of China was screened for anti-cancer activities. Three limonoid compounds, termed A1541-43, originally isolated from the plant Melia azedarach, exhibiting strong anti-leukemic activity. The anti-neoplastic activity and the biological target of these compounds were explored using various methods, including western blotting, flow cytometry, molecular docking and animal model for leukemia. RESULTS: Compounds A1541-43, exhibiting potent anti-leukemic activity, was shown to induce ERK1/2 phosphorylation. In contrast, the natural product Cedrelone, which shares structural similarities with A1541-43, functions as a potent inhibitor of ERK1/2. We provided evidence that A1541-43 and Cedrelone specifically target ERK1/2, but not the upstream MAPK/ERK pathway. Computational docking analysis predicts that compounds A1541-43 bind a region in ERK1/2 that is distinct from that to which Cedrelone and EQW bind. Interestingly, both A1541-43, which act as ERK1/2 agonists, and Cedrelone, which inhibit these kinases, exerted strong anti-proliferative activity against multiple leukemic cell lines, and induced robust apoptosis as well as erythroid and megakaryocytic differentiation in erythroleukemic cell lines. These compounds also suppressed tumor progression in a mouse model of erythroleukemia. CONCLUSIONS: This study identifies for the first time activators of ERK1/2 with therapeutic potential for the treatment of cancers driven by dysregulation of the MAPK/ERK pathway and possibly for other disorders.

blaCTX-M-1/9/1 Hybrid Genes May Have Been Generated from blaCTX-M-15 on an IncI2 Plasmid.

Three hybrid CTX-M ß-lactamases, CTX-M-64, CTX-M-123, and CTX-M-132, with N and C termini matching CTX-M-1 group enzymes and centers matching CTX-M-9 group enzymes, have been identified. The hybrid gene sequences suggested recombination between blaCTX-M-15 and blaCTX-M-14, the two most common blaCTX-M variants worldwide. However, blaCTX-M-64 and blaCTX-M-123 are found in an ISEcp1-blaCTX-M transposition unit with a 45-bp "spacer," rather than the 48 bp usually associated with blaCTX-M-15, and 112 bp of IncA/C plasmid backbone. This is closer to the context of blaCTX-M-55, which has one nucleotide difference from blaCTX-M-15, on IncI2 plasmid pHN1122-1. Here, we characterized an IncI2 plasmid carrying blaCTX-M-15 with a 45-bp spacer (pHNY2-1) by complete sequencing and also sequenced IncI2 plasmids carrying blaCTX-M-64 (pHNAH46-1) or blaCTX-M-132 (pHNLDH19) and an IncI1 plasmid carrying blaCTX-M-123 (pHNAH4-1). pHNY2-1 has the same ISEcp1-blaCTX-M-IncA/C insertion as pHN1122-1, pHNAH46-1, and pHNLDH19, and all four plasmid backbones are almost identical. pHNAH4-1 (IncI1 sequence type 108 [ST108]) carries a transposition unit that includes a 2,720-bp fragment of the IncI2 backbone, suggesting ISEcp1-mediated transfer of blaCTX-M-IncA/C-IncI2 to an IncI1 plasmid. All three hybrid blaCTX-M genes may have resulted from recombination between blaCTX-M-14 and blaCTX-M-15 with a 45-bp spacer on an IncI2 plasmid. Five additional Escherichia coli isolates of different sequence types from different provinces, farms, and/or animals had blaCTX-M-64 on a pHNAH46-1-like IncI2 plasmid and 9 had blaCTX-M-123 on a pHNAH4-1-like IncI1 ST108 plasmid. Thus, epidemic IncI plasmids may be responsible for the spread of blaCTX-M-64 and blaCTX-M-123 between different animals and different locations in China.

An intricate regulatory circuit between FLI1 and GATA1/GATA2/LDB1/ERG dictates erythroid vs. megakaryocytic differentiation.

During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP­like cell line HEL western blotting, RT­qPCR, lentivirus­mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation­specific (ETS) transcription factor friend leukemia integration factor 1 (Fli­1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1­mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation­specific­related gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA­mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.

B4 suppresses lymphoma progression by inhibiting fibroblast growth factor binding protein 1 through intrinsic apoptosis.

Lymphoma positions as the fifth most common cancer, in the world, reporting remarkable deaths every year. Several promising strategies to counter this disease recently include utilizing small molecules that specifically target the lymphoma cellular proteins to overwhelm its progression. FGFBP1 is a soluble intracellular protein that progresses cancer cell proliferation and is upregulated in several cancers. Therefore, inhibiting FGFBP1 could significantly slow down lymphoma progression through triggering apoptosis. Thus, in this study, a flavonoid B4, isolated from Cajanus cajan, has been investigated for its effects of B4 on lymphoma, specifically as an FGFBP1 inhibitor. B4 could selectively hinder the growth of lymphoma cells by inducing caspase-dependent intrinsic apoptosis through G1/S transition phase cell cycle arrest. RNA sequencing analysis revealed that B4 regulates the genes involved in B-cell proliferation and DNA replication by inhibiting FGFBP1 in vitro. B4 increases the survival rate of lymphoma mice. B4 also represses the growth of patient-derived primary lymphoma cells through FGFBP1 inhibition. Drug affinity responsive target stability experimentations authorize that B4 powerfully binds to FGFBP1. The overexpression of FGFBP1 raises the pharmacological sensitivity of B4, supplementing its specific action on lymphoma cells. This study pioneers the estimation of B4 as a possible anticancer agent for lymphoma treatment. These outcomes highlight its selective inhibitory effects on lymphoma cell growth by downregulating FGFBP1 expression through intrinsic apoptosis, causing mitochondrial and DNA damage, ultimately leading to the inhibition of lymphoma progression. These suggest B4 may be a novel FGFBP1 inhibitor for the lymphoma treatment.

FLI1 Regulates Histamine Decarboxylase Expression to Control Inflammation Signaling and Leukemia Progression.

Aim: Histamine decarboxylase (HDC) catalyzes decarboxylation of histidine to generate histamine. This enzyme affects several biological processes including inflammation, allergy, asthma, and cancer, although the underlying mechanism is not fully understood. The present study provides a novel insight into the relationship between the transcription factor FLI1 and its downstream target HDC, and their effects on inflammation and leukemia progression. Methods: Promoter analysis combined with chromatin immunoprecipitation (ChIp) was used to demonstrate binding of FLI1 to the promoter of HDC in leukemic cells. Western blotting and RT-qPCR were used to determine expression of HDC and allergy response genes, and lentivirus shRNA was used to knock-down target genes. Proliferation, cell cycle, apoptosis assays and molecular docking were used to determine the effect of HDC inhibitors in culture. An animal model of leukemia was employed to test the effect of HDC inhibitory compounds in vivo. Results: Results presented herein demonstrate that FLI1 transcriptionally regulates HDC by direct binding to its promoter. Using genetic and pharmacological inhibition of HDC, or the addition of histamine, the enzymatic product of HDC, we show neither have a discernable effect on leukemic cell proliferation in culture. However, HDC controls several inflammatory genes including IL1B and CXCR2 that may influence leukemia progression in vivo through the tumor microenvironment. Indeed, diacerein, an IL1B inhibitor, strongly blocked Fli-1-induced leukemia in mice. In addition to allergy, FLI1 is shown to regulate genes associated with asthma such as IL1B, CPA3 and CXCR2. Toward treatment of these inflammatory conditions, epigallocatechin (EGC), a tea polyphenolic compound, is found strongly inhibit HDC independently of FLI1 and its downstream effector GATA2. Moreover, the HDC inhibitor, tetrandrine, suppressed HDC transcription by directly binding to and inhibiting the FLI1 DNA binding domain, and like other FLI1 inhibitors, tetrandrine strongly suppressed cell proliferation in culture and leukemia progression in vivo. Conclusion: These results suggest a role for the transcription factor FLI1 in inflammation signaling and leukemia progression through HDC and point to the HDC pathway as potential therapeutics for FLI1-driven leukemia.

An unusual indole-diterpenoid with C-17 norcassane skeleton from Euphorbia fischeriana induces HEL cell cycle arrest and apoptosis.

Two new polycyclic diterpenoids, euphkanoids H and I (1 and 2), along with 6 known analogues (2-8) were isolated from the roots of Euphorbia fischeriana, a traditional Chinese medicine. Their structures were identified by spectral methods, and the absolute configurations of 1 and 2 were determined by ECD calculation and single crystal X-ray diffraction, respectively. Compound 1 represents the first example of C-17 norcassane indole-diterpenes. All the isolates were screened for antiproliferative activity against a panel of human cancer cell lines using the MTT assay, and 1 showed significant cytotoxicity against HEL cells (IC50 = 3.2 µM). Simple mechanistic study revealed that 1 could induce cell cycle arrest at G0/G1 phase and apoptosis in HEL cells.

A critical ETV4/Twist1/Vimentin axis in Ha-RAS-induced aggressive breast cancer.

RAS oncogenes are major drivers of diverse types of cancer. However, they are largely not druggable, and therefore targeting critical downstream pathways and dependencies is an attractive approach. We have isolated a tumorigenic cell line (FE1.2), which exhibits mesenchymal characteristics, after inoculating Ha-Ras-expressing retrovirus into mammary glands of rats, and subsequently isolated a non-aggressive revertant cell line (FC5). This revertant has lost the rat Ha-Ras driver and showed a more epithelial morphology, slower proliferation in culture, and reduced tumorigenicity in vivo. Re-expression of human Ha-RAS in these cells (FC5-RAS) reinduced mesenchymal morphology, higher proliferation rate, and tumorigenicity that was still significantly milder than parental FE1.2 cells. RNA-seq analysis of FC5-RAS vs FC5-Vector cells identified multiple genes whose expressions were regulated by Ha-RAS. This analysis also identified many genes including those controlling cell growth whose expression was altered by loss of HA-Ras in FC5 cells but remained unchanged upon reintroduction of Ha-RAS. These results suggest that targeting the Ha-Ras driver oncogene induces partial tumor regression, but it still denotes strong efficacy for cancer therapy. Among the RAS-responsive genes, we identified Twist1 as a critical mediator of epithelial-to-mesenchymal transition through the direct transcriptional regulation of vimentin. Mechanistically, we show that Twist1 is induced by the ETS gene, ETV4, downstream of Ha-RAS, and that inhibition of ETV4 suppressed the growth of breast cancer cells driven by the Ha-RAS pathway. Targeting the ETV4/Twist1/Vimentin axis may therefore offer a therapeutic modality for breast tumors driven by the Ha-RAS pathway.

FLI1 regulates inflammation-associated genes to accelerate leukemogenesis.

Inflammation plays a critical role in cancer initiation and progression, and is induced by inflammatory factors that are direct target of oncogenes and tumor suppressors. The ETS related transcription factor Fli-1 is involved in the induction and progression of various cancers; yet its role in inflammation is not well-defined. Using RNAseq analysis, we herein demonstrate that FLI1 induces the inflammatory pathway in erythroleukemia cells. Majority of genes within the TNF signaling pathway including TNF and IL1B were identified as transcriptional targets of FLI1. TNF expression is indirectly regulated by FLI1 through upregulation of another ETS related oncogene, SPI1/PU.1. Pharmacological inhibition of TNF significantly inhibited leukemia cell proliferation in culture. In contrast, IL1B expression is directly regulated by FLI1 through promoter binding and transcriptional activation. The secreted factor IL1B binds its canonical receptors to accelerate cancer progression through changes in the surrounding tumor microenvironment, fostering cell survival, proliferation and migration. Through network analysis, we identified IL1B-interacting genes whose expression is also regulated by FLI1. Among these, IL1B-interacting proteins, FOS, JUN, JUNB and CASP1 are negatively regulated by FLI1. Treatment of leukemia cells with inhibitors of AP1 (TAN IIA) and CASP1 (765VX) significantly accelerated FLI1-dependent leukemia progression. These results emphasize the significance of FLI1 in regulating the inflammatory pathway. Targeting these inflammatory genes downstream of FLI1 offers a novel strategy to treat leukemic progression associated with overexpression of this oncogenic ETS transcription factor.

Stilbene B10 induces apoptosis and tumor suppression in lymphoid Raji cells by BTK-mediated regulation of the KRAS/HDAC1/EP300/PEBP1 axis.

Lymphoma is a cancer of the lymphoid cells that originated in matured B or T cells. The bioactive natural compounds can efficiently treat this disease with lesser side effects. Thus, in this study, a natural stilbene B10 (3-methoxy 5-hydroxy stilbene) isolated from Cajanus cajan (Pigeon Pea) was screened for its anti-proliferative efficacy against 13 cancer cell lines. B10 showed a potential effect on the human lymphoma (Raji) cells. Cytotoxicity analysis of B10 has revealed IC50 concentrations in Raji cells at low doses (18 µM) than other cancer cell lines. The B10 could significantly cause dose and time-dependent inhibition in the proliferation of Raji cells triggering intrinsic apoptosis and S/G1 phase cellular arrest. There was an increased expression of phospho-γ-H2A.X and decreased expression of cyclin D1, causing DNA damage and cell cycle arrest, post- B10 treatments. The mitochondrial membrane potential (MMP) variations observed after B10 treatment led to changes in Bax/Bcl-2 ratio, cytochrome C release, and enhanced expression of cleaved caspase3, 9, PARP-1, and APAF-1. The B10 inhibited the proliferation of Raji cells by significantly downregulating the expression of KRAS, BTK, MDM2, P-JAK2, P-STAT3, PI3K, HDAC1/2, SIRT7, and EP300. The treatment upregulated the tumor suppressor genes PEBP1 and SAP18. Thus, the study could reveal the selective inhibitory effects of B10 on lymphoma, suggesting it as a probable innovative chemotherapeutic agent.

UM171 cooperates with PIM1 inhibitors to restrict HSC expansion markers and suppress leukemia progression.

The pyrimido-indole derivative UM171 promotes human Hematopoietic Stem Cells Expansion (HSCE), but its impact on leukemia is not known. Herein, we show in a mouse model of erythroleukemia that UM171 strongly suppresses leukemia progression. UM171 inhibits cell cycle progression and apoptosis of leukemic cells in culture. The effect of UM171 on leukemia differentiation was accompanied by increased expression of HSCE markers. RNAseq analysis combined with Q-RT-PCR and western blotting revealed that the PIM1 protein kinase is highly elevated in response to UM171 treatment. Moreover, docking analysis combined with immunoprecipitation assays revealed high binding affinity of UM171 to PIM1. Interestingly, pan-PIM kinase inhibitors counteracted the effect of UM171 on HSCE marker expression and PIM1 transcription, but not its suppression of leukemic cell growth. Moreover, combination treatment with UM171 and a pan-PIM inhibitor further suppressed leukemic cell proliferation compared to each drug alone. To uncover the mechanism of growth inhibition, we showed strong upregulation of the cyclin-dependent kinase inhibitor P21CIP1 and the transcription factor KLF2 by UM171. In accordance, KLF2 knockdown attenuated growth inhibition by UM171. KLF2 upregulation by UM171 is also responsible for the activation of P21CIP1 in leukemic cells leading to a G1/S arrest and suppression of leukemogenesis. Thus, suppression of leukemic growth by UM171 through KLF2 and P21CIP1 is thwarted by PIM-mediated expansion of leukemic stemness, uncovering a novel therapeutic modality involving combined UM171 plus PIM inhibitors.

Garmultin-A Incites Apoptosis in CB3 Cells Through miR-17-5p by Attenuating Poly (ADP-Ribose) Polymerase-1.

Background: Leukemia accounts for a large number of deaths, worldwide, every year. Treating this ailment is always a challenging job. Recently, oncogenic miRNA leading to apoptosis are highly promising targets of many natural products. In this study, Garmultin-A (GA), isolated from the bark of Garcinia multiflora, was elucidated for its anti-leukemic effect in CB3 cells. Methods: The effect of the compound on CB3 cell viability was detected by MTT assay and apoptosis by FITC Annexin V/PI and Hochest 33258 staining. The western blot analysis assessed the BAX, BCL2, cMYC, pERK, and PARP-1 protein levels. Autodock analysis predicted the ligand-protein interactions. q-RT-PCR quantified the miR-17-5p expression. Luciferase assay confirmed the interaction between PARP-1 and miR-17-5p. Results: We uncover that GA leads to apoptosis by inducing overexpression of miR-17-5p and significantly downregulate PARP-1 protein levels in CB3 cells. The overexpression of miR-17-5p promotes apoptosis, and the miR-17-5p antagomirs restore GA-triggered apoptosis. Notably, we disclose that PARP-1 is a direct target of miR-17-5p. Increased pro-apoptotic and reduced anti-apoptosis protein levels were also observed in GA-treated CB3 cells. Conclusion: These results provide critical insights that GA could induce apoptosis in CB3 cells through targeting miR-17-5p by attenuating PARP-1. Thus, GA could act as a novel therapeutic agent for erythroleukemia.

FLI1 accelerates leukemogenesis through transcriptional regulation of pyruvate kinase-L/R and other glycolytic genes.

In cancer cells, multiple oncogenes and tumor suppressors control glycolysis to sustain rapid proliferation. The ETS-related transcription factor Fli1 plays a critical role in the induction and progression of leukemia, yet, the underlying mechanism of this oncogenic event is still not fully understood. In this study, RNAseq analysis of FLI1-depleted human leukemic cells revealed transcriptional suppression of the PKLR gene and activation of multiple glycolytic genes, such as PKM1/2. Pharmacological inhibition of glycolysis by PKM2 inhibitor, Shikonin, significantly suppressed leukemic cell proliferation. FLI1 directly binds to the PKLR promoter, leading to the suppression of this inhibitor of glycolysis. In accordance, shRNA-mediated depletion of PKLR in leukemic HEL cells expressing high levels of FLI1 accelerated leukemia proliferation, pointing for the first time to its tumor suppressor function. PKLR knockdown also led to downregulation of the erythroid markers EPOR, HBA1, and HBA2 and suppression of erythroid differentiation. Interestingly, silencing of PKLR in HEL cells significantly increased FLI1 expression, which was associated with faster proliferation in culture. In FLI1-expressing leukemic cells, lower PKLR expression was associated with higher expression of PKM1 and PKM2, which promote aerobic glycolysis. Finally, injection of pyruvate, a known inhibitor of glycolysis, into leukemia mice significantly suppressed leukemogenesis. These results demonstrate that FLI1 promotes leukemia in part by inducing glycolysis, implicates PKLR in erythroid differentiation, and suggests that targeting glycolysis may be an attractive therapeutic strategy for cancers driven by FLI1 overexpression.

FLI1 Induces Megakaryopoiesis Gene Expression Through WAS/WIP-Dependent and Independent Mechanisms; Implications for Wiskott-Aldrich Syndrome.

Wiskott-Aldrich Syndrome, WAS/WAVE, is a rare, X-linked immune-deficiency disease caused by mutations in the WAS gene, which together with its homolog, N-WASP, regulates actin cytoskeleton remodeling and cell motility. WAS patients suffer from microthrombocytopenia, characterized by a diminished number and size of platelets, though the underlying mechanism is not fully understood. Here, we identified FLI1 as a direct transcriptional regulator of WAS and its binding partner WIP. Depletion of either WAS or WIP in human erythroleukemic cells accelerated cell proliferation, suggesting tumor suppressor function of both genes in leukemia. Depletion of WAS/WIP also led to a significant reduction in the percentage of CD41 and CD61 positive cells, which mark committed megakaryocytes. RNAseq analysis revealed common changes in megakaryocytic gene expression following FLI1 or WASP knockdown. However, in contrast to FLI1, WASP depletion did not alter expression of late-stage platelet-inducing genes. N-WASP was not regulated by FLI1, yet its silencing also reduced the percentage of CD41+ and CD61+ megakaryocytes. Moreover, combined knockdown of WASP and N-WASP further suppressed megakaryocyte differentiation, indicating a major cooperation of these related genes in controlling megakaryocytic cell fate. However, unlike WASP/WIP, N-WASP loss suppressed leukemic cell proliferation. WASP, WIP and N-WASP depletion led to induction of FLI1 expression, mediated by GATA1, and this may mitigate the severity of platelet deficiency in WAS patients. Together, these results uncover a crucial role for FLI1 in megakaryocyte differentiation, implicating this transcription factor in regulating microthrombocytopenia associated with Wiskott-Aldrich syndrome.