The HOX11/TLX1 (hereafter referred to as HOX11) homeobox gene was originally identified at a t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T cell acute lymphoblastic leukemias (T-ALLs). We previously reported a predisposition to aberrant spindle assembly checkpoint arrest and heightened incidences of chromosome missegregation in HOX11-overexpressing B lymphocytes following exposure to spindle poisons. The purpose of the current study was to evaluate cell cycle specific expression of HOX11.

The ETS transcription factors play a critical role during hematopoiesis. In F-MuLV-induced erythroleukemia, Fli‑1 insertional activation producing high expression of this transcription factor required to promote proliferation. How deregulated Fli‑1 expression alters the balance between erythroid differentiation and proliferation is unknown. To address this issue, we exogenously overexpressed Fli‑1 in an erythroleukemic cell harboring activation of spi‑1/PU.1, another ETS gene involved in erythroleukemogenesis. While the proliferation in culture remains unaffected, Fli‑1 overexpression imparts morphological and immunohistochemical characteristics of immature erythroid progenitors. Fli‑1 overexpression in erythroleukemic cells increased the numbers of erythroid colonies on methylcellulose and reduced tumorigenicity as evidenced by increase latency of erythroleukemogenesis in mice inoculated with these cells. Although all transplanted mice developed enlargement of the spleen and liver due to leukemic infiltration, Fli‑1 overexpression altered the hematopoietic phenotype, significantly increasing the expression of regulatory hematopoietic genes cKIT, SCA-1, CD41 and CD71. In contrast, expression of Spi‑1/PU.1 in a Fli‑1 producing erythroleukemia cell line in which fli‑1 is activated, resulted in increased proliferation through activation of growth promoting proteins MAPK, AKT, cMYC and JAK2. Importantly, these progenitors express high levels of markers such as CD71 and TER119 associated with more mature erythroid cells. Thus, Fli‑1 overexpression induces a de-differentiation program by reverting CFU-E to BFU-E erythroid progenitor activity, while Spi‑1/PU.1 promoting maturation from BFU-E to CFU-E.

The friend leukemia integration 1 (Fli-1) gene is involved in the expression control of key genes in multiple pathogenic/physiological processes, including cell growth, differentiation, and apoptosis; this implies that Fli-1 is a strong candidate for drug development. In our previous study, a 3',5'-diprenylated chalcone, (E)-1-(2-hydroxy-4-methoxy-3,5-diprenyl) phenyl-3-(3-pyridinyl)-propene-1-one (C10), was identified as a novel anti-prostate cancer (PCa) agent. Here, we investigated the molecular mechanisms underlying the anti-cancer effects of C10 on the growth, metastasis, and invasion of PC3 cells in vitro. Our results show that C10 exhibited a strong inhibitory effect on proliferation and metastasis of PC3 cells via several cellular and flow cytometric analyses. Further mechanism studies revealed that C10 likely serves as an Fli-1 agonist for regulating the expression of Fli-1 target genes including phosphatidylinositol 3-kinase (P110), murine double minute2 (MDM2), B-cell lymphoma-2 (Bcl-2), Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP-1), and globin transcription factor-1 (Gata-1) as well as the phosphorylation of extracellular-regulated protein kinases 1 (ERK1). Further, we confirmed that C10 can regulate the expressions of vascular endothelial growth factor 1 (VEGF-1), transforming growth factor-β2 (TGF-β2), intercellular cell adhesion molecule-1 (ICAM-1), p53, and matrix metalloproteinase 1 (MMP-1) genes associated with tumor apoptosis, migration, and invasion. Thus, C10 exhibits stronger anticancer activity with novel molecular targets and regulatory molecular mechanisms, indicating its great potency for development as a novel targeted anticancer drug.

Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic β-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.

Graft-versus-host disease (GVHD), manifesting as either acute (aGVHD) or chronic (cGVHD), presents significant life-threatening complications following allogeneic hematopoietic cell transplantation. Here, we investigated Friend virus leukemia integration 1 (Fli-1) in GVHD pathogenesis and validated Fli-1 as a therapeutic target. Using genetic approaches, we found that Fli-1 dynamically regulated different T cell subsets in allogeneic responses and pathogenicity in the development of aGVHD and cGVHD. Compared with homozygous Fli1-deficient or WT T cells, heterozygous Fli1-deficient T cells induced the mildest GVHD, as evidenced by the lowest Th1 and Th17 cell differentiation. Single-cell RNA-Seq analysis revealed that Fli-1 differentially regulated CD4+ and CD8+ T cell responses. Fli-1 promoted the transcription of Th1/Th17 pathways and T cell receptor-inducible (TCR-inducible) transcription factors in CD4+ T cells, while suppressing activation- and function-related gene pathways in CD8+ T cells. Importantly, a low dose of camptothecin, topotecan, or etoposide acted as a potent Fli-1 inhibitor and significantly attenuated GVHD severity, while preserving the graft-versus-leukemia (GVL) effect. This observation was extended to a xenograft model, in which GVHD was induced by human T cells. In conclusion, we provide evidence that Fli-1 plays a crucial role in alloreactive CD4+ T cell activation and differentiation and that targeting Fli-1 may be an attractive strategy for treating GVHD without compromising the GVL effect.

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.

Acute erythro- and megakaryoblastic leukaemias are associated with very poor prognoses and the mechanism of blastic transformation is insufficiently elucidated. The murine Graffi leukaemia retrovirus induces erythro- and megakaryoblastic leukaemias when inoculated into NFS mice and represents a good model to study these leukaemias.

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) encodes a tumor suppressor gene implicated in the growth of various tumor types including breast cancer. We previously demonstrated that recombinant adenovirus-mediated mda-7/IL-24 expression in the mammary glands of carcinogen-treated (methylnitrosourea, MNU) rats suppressed mammary tumor development. Since most MNU-induced tumors in rats contain activating mutations in Ha-ras, which arenot frequently detected in humans, we presently examined the effect of MDA-7/IL-24 on Her2/Neu-induced mammary tumors, in which the RAS pathway is induced. We generated tet-inducible MDA-7/IL-24 transgenic mice and crossed them with Her2/Neu transgenic mice. Triple compound transgenic mice treated with doxycycline exhibited a strong inhibition of tumor development, demonstrating tumor suppressor activity by MDA-7/IL-24 in immune-competent mice. MDA-7/IL-24 induction also inhibited growth of tumors generated following injection of Her2/Neu tumor cells isolated from triple compound transgenic mice that had not been treated with doxycycline, into the mammary fat pads of isogenic FVB mice. Despite initial growth suppression, tumors in triple compound transgenic mice lost mda-7/IL-24 expression and grew, albeit after longer latency, indicating that continuous presence of this cytokine within tumor microenvironment is crucial to sustain tumor inhibitory activity. Mechanistically, MDA-7/IL-24 exerted its tumor suppression effect on HER2+ breast cancer cells, at least in part, through PERP, a member of PMP-22 family with growth arrest and apoptosis-inducing capacity. Overall, our results establish mda-7/IL-24 as a suppressor of mammary tumor development and provide a rationale for using this cytokine in the prevention/treatment of human breast cancer.

The disruption of protein translation machinery is a common feature of cancer initiation and progression, and drugs that target protein translation offer new avenues for therapy. The translation initiation factor, eukaryotic initiation factor 4E (eIF4E), is induced in a number of cancer cell lines and is one such candidate for therapeutic intervention. Friend leukemia integration 1 (FLI1) is a potent oncogenic transcription factor that promotes various types of cancer by promoting several hallmarks of cancer progression. FLI1 has recently been implicated in protein translation through yet unknown mechanisms. This study identified a positive association between FLI1 expression and mitogen‑activated protein kinase (MAPK)‑interacting serine/threonine kinase1 (MKNK1), the immediate upstream regulator of the eIF4E initiation factor. The short hairpin RNA (shRNA)‑mediated silencing or overexpression of FLI1 in leukemic cell lines downregulated or upregulated MKNK1 expression, respectively. Promoter analysis identified a potent FLI1 binding site in the regulatory region of the MKNK1 promoter. In transient transfection experiments, FLI1 increased MKNK1 promoter activity, which was blocked by mutating the FLI1 binding site. FLI1 specifically affected the expression of MKNK1, but not that of MKNK2. The siRNA‑mediated downregulation of MKNK1 downregulated the expression of survivin (BIRC5) and significantly suppressed cell proliferation in culture. FLI1 inhibitory compounds were shown to downregulate this oncogene through the suppression of MAPK/extracellular‑regulated kinase (ERK) signaling and the subsequent activation of miR‑145, leading to a lower MKNK1 expression and the suppression of leukemic growth. These results uncover a critical role for FLI1 in the control of protein translation and the importance of targeting its function and downstream mediators, such as MKNK1, for cancer therapy.

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.

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.

The eukaryotic elongation factor-2 kinase, eEF2K, which restricts protein translation elongation, has been identified as a potential therapeutic target for diverse types of malignancies including triple negative breast cancer (TNBC). However, the contexts in which eEF2K inhibition is essential in TNBC and its consequences on the proteome are largely unknown. Here we show that genetic or pharmacological inhibition of eEF2K cooperated with glutamine (Gln) starvation, and synergized with glutaminase (GLS1) inhibitors to suppress growth of diverse TNBC cell lines. eEF2K inhibition also synergized with depletion of eukaryotic translation initiation factor 4E-binding protein 1 (eIF4EBP1; 4EBP1), a suppressor of eukaryotic protein translation initiation factor 4E (eIF4E), to induce c-MYC and Cyclin D1 expression, yet attenuate growth of TNBC cells. Proteomic analysis revealed that whereas eEF2K depletion alone uniquely induced Cyclin Dependent Kinase 1 (CDK1) and 6 (CDK6), combined depletion of eEF2K and 4EBP1 resulted in overlapping effects on the proteome, with the highest impact on the 'Collagen containing extracellular matrix' pathway (e.g. COL1A1), as well as the amino-acid transporter, SLC7A5/LAT1, suggesting a regulatory loop via mTORC1. In addition, combined depletion of eEF2K and 4EBP1 indirectly reduced the levels of IFN-dependent innate immune response-related factors. Thus, eEF2K inhibition triggers cell cycle arrest/death under unfavourable metabolic conditions such as Gln-starvation/GLS1 inhibition or 4EBP1 depletion, uncovering new therapeutic avenues for TNBC and underscoring a pressing need for clinically relevant eEF2K inhibitors.

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.

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.

CDK4/6 inhibitors are effective against cancer cells expressing the tumor suppressor RB1, but not RB1-deficient cells, posing the challenge of how to target RB1 loss. In triple-negative breast cancer (TNBC), RB1 and PTEN are frequently inactivated together with TP53. We performed kinome/phosphatase inhibitor screens on primary mouse Rb/p53-, Pten/p53-, and human RB1/PTEN/TP53-deficient TNBC cell lines and identified CDC25 phosphatase as a common target. Pharmacological or genetic inhibition of CDC25 suppressed growth of RB1-deficient TNBC cells that are resistant to combined CDK4/6 plus CDK2 inhibition. Minimal cooperation was observed in vitro between CDC25 antagonists and CDK1, CDK2, or CDK4/6 inhibitors, but strong synergy with WEE1 inhibition was apparent. In accordance with increased PI3K signaling following long-term CDC25 inhibition, CDC25 and PI3K inhibitors effectively synergized to suppress TNBC growth both in vitro and in xenotransplantation models. These results provide a rationale for the development of CDC25-based therapies for diverse RB1/PTEN/TP53-deficient and -proficient TNBCs.

The ETS transcription factor Fli-1 controls the expression of genes involved in hematopoiesis including cell proliferation, survival, and differentiation. Dysregulation of Fli-1 induces hematopoietic and solid tumors, rendering it an important target for therapeutic intervention. Through high content screens of a library of chemicals isolated from medicinal plants in China for inhibitors of a Fli-1 transcriptional reporter cells, we hereby report the identification of diterpenoid-like compounds that strongly inhibit Fli-1 transcriptional activity. These agents suppressed the growth of erythroleukemic cells by inducing apoptosis and differentiation. They also inhibited survival and proliferation of B-cell leukemic cell lines as well as primary B-cell lymphocytic leukemia (B-CLL) isolated from 7 patients. Moreover, these inhibitors blocked leukemogenesis in a mouse model of erythroleukemia, in which Fli-1 is the driver of tumor initiation. Computational docking analysis revealed that the diterpenoid-like compounds bind with high affinity to nucleotide residues in a pocket near the major groove within the DNA-binding sites of Fli-1. Functional inhibition of Fli-1 by these compounds triggered its further downregulation through miR-145, whose promoter is normally repressed by Fli-1. These results uncover the importance of Fli-1 in leukemogenesis, a Fli-1-miR145 autoregulatory loop and new anti-Fli-1 diterpenoid agents for the treatment of diverse hematological malignancies overexpressing this transcription factor.

The ETS-related transcription factor Fli-1 affects many developmental programs including erythroid and megakaryocytic differentiation, and is frequently de-regulated in cancer. Fli-1 was initially isolated following retrovirus insertional mutagenesis screens for leukemic initiator genes, and accordingly, inhibition of this transcription factor can suppress leukemia through induction of erythroid differentiation. To search for modulators of Fli-1, we hereby performed repurposing drug screens with compounds isolated from Chinese medicinal plants. We identified agents that can transcriptionally activate or inhibit a Fli-1 reporter. Remarkably, agents that increased Fli-1 transcriptional activity conferred a strong anti-cancer activity upon Fli-1-expressing leukemic cells in culture. As opposed to drugs that suppress Fli1 activity and lead to erythroid differentiation, growth suppression by these new Fli-1 transactivating compounds involved erythroid to megakaryocytic conversion (EMC). The identified compounds are structurally related to diterpene family of small molecules, which are known agonists of protein kinase C (PKC). In accordance, these PKC agonists (PKCAs) induced PKC phosphorylation leading to activation of the mitogen-activated protein kinase (MAPK) pathway, increased cell attachment and EMC, whereas pharmacological inhibition of PKC or MAPK diminished the effect of our PKCAs. Moreover, in a mouse model of leukemia initiated by Fli-1 activation, the PKCA compounds exhibited strong anti-cancer activity, which was accompanied by increased presence of CD41/CD61 positive megakaryocytic cells in leukemic spleens. Thus, PKC agonists offer a novel approach to combat Fli-1-induced leukemia, and possibly other cancers,by inducing EMC in part through over-activation of the PKC-MAPK-Fli-1 pathway.

Pineoblastoma is a rare pediatric cancer induced by germline mutations in the tumor suppressors RB1 or DICER1. Presence of leptomeningeal metastases is indicative of poor prognosis. Here we report that inactivation of Rb plus p53 via a WAP-Cre transgene, commonly used to target the mammary gland during pregnancy, induces metastatic pineoblastoma resembling the human disease with 100% penetrance. A stabilizing mutation rather than deletion of p53 accelerates metastatic dissemination. Deletion of Dicer1 plus p53 via WAP-Cre also predisposes to pineoblastoma, albeit with lower penetrance. In silico analysis predicts tricyclic antidepressants such as nortriptyline as potential therapeutics for both pineoblastoma models. Nortriptyline disrupts the lysosome, leading to accumulation of non-functional autophagosome, cathepsin B release and pineoblastoma cell death. Nortriptyline further synergizes with the antineoplastic drug gemcitabine to effectively suppress pineoblastoma in our preclinical models, offering new modality for this lethal childhood malignancy.

Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFβ and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.

Immune-checkpoint (IC) modulators like the poliovirus receptor (PVR) and programmed death ligand 1 (PD-L1) attenuate innate and adaptive immune responses and are potential therapeutic targets for diverse malignancies, including triple-negative breast cancer (TNBC). The retinoblastoma tumor suppressor, pRB, controls cell growth through E2F1-3 transcription factors, and its inactivation drives metastatic cancer, yet its effect on IC modulators is contentious. Here, we show that RB-loss and high E2F1/E2F2 signatures correlate with expression of PVR, CD274 (PD-L1 gene) and other IC modulators and that pRB represses whereas RB depletion and E2F1 induce PVR and CD274 in TNBC cells. Accordingly, the CDK4/6 inhibitor, palbociclib, suppresses both PVR and PD-L1 expression. Palbociclib also counteracts the effect of CDK4 on SPOP, leading to its depletion, but the overall effect of palbociclib is a net reduction in PD-L1 level. Hydrochloric acid, commonly used to solubilize palbociclib, counteracts its effect and induces PD-L1 expression. Remarkably, lactic acid, a by-product of glycolysis, also induces PD-L1 as well as PVR. Our results suggest a model in which CDK4/6 regulates PD-L1 turnover by promoting its transcription via pRB-E2F1 and degradation via SPOP and that the CDK4/6-pRB-E2F pathway couples cell proliferation with the induction of multiple innate and adaptive immunomodulators, with direct implications for cancer progression, anti-CDK4/6- and IC-therapies.