Therapeutic targeting of EP300/CBP by bromodomain inhibition in hematologic malignancies.

CCS1477 (inobrodib) is a potent, selective EP300/CBP bromodomain inhibitor which induces cell-cycle arrest and differentiation in hematologic malignancy model systems. In myeloid leukemia cells, it promotes rapid eviction of EP300/CBP from an enhancer subset marked by strong MYB occupancy and high H3K27 acetylation, with downregulation of the subordinate oncogenic network and redistribution to sites close to differentiation genes. In myeloma cells, CCS1477 induces eviction of EP300/CBP from FGFR3, the target of the common (4; 14) translocation, with redistribution away from IRF4-occupied sites to TCF3/E2A-occupied sites. In a subset of patients with relapsed or refractory disease, CCS1477 monotherapy induces differentiation responses in AML and objective responses in heavily pre-treated multiple myeloma. In vivo preclinical combination studies reveal synergistic responses to treatment with standard-of-care agents. Thus, CCS1477 exhibits encouraging preclinical and early-phase clinical activity by disrupting recruitment of EP300/CBP to enhancer networks occupied by critical transcription factors.

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway.

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

HMG20B stabilizes association of LSD1 with GFI1 on chromatin to confer transcription repression and leukemia cell differentiation block.

Pharmacologic inhibition of LSD1 induces molecular and morphologic differentiation of blast cells in acute myeloid leukemia (AML) patients harboring MLL gene translocations. In addition to its demethylase activity, LSD1 has a critical scaffolding function at genomic sites occupied by the SNAG domain transcription repressor GFI1. Importantly, inhibitors block both enzymatic and scaffolding activities, in the latter case by disrupting the protein:protein interaction of GFI1 with LSD1. To explore the wider consequences of LSD1 inhibition on the LSD1 protein complex we applied mass spectrometry technologies. We discovered that the interaction of the HMG-box protein HMG20B with LSD1 was also disrupted by LSD1 inhibition. Downstream investigations revealed that HMG20B is co-located on chromatin with GFI1 and LSD1 genome-wide; the strongest HMG20B binding co-locates with the strongest GFI1 and LSD1 binding. Functional assays demonstrated that HMG20B depletion induces leukemia cell differentiation and further revealed that HMG20B is required for the transcription repressor activity of GFI1 through stabilizing LSD1 on chromatin at GFI1 binding sites. Interaction of HMG20B with LSD1 is through its coiled-coil domain. Thus, HMG20B is a critical component of the GFI1:LSD1 transcription repressor complex which contributes to leukemia cell differentiation block.

STAT3 promotes melanoma metastasis by CEBP-induced repression of the MITF pathway.

Metastatic melanoma is hallmarked by its ability of phenotype switching to more slowly proliferating, but highly invasive cells. Here, we tested the impact of signal transducer and activator of transcription 3 (STAT3) on melanoma progression in association with melanocyte inducing transcription factor (MITF) expression levels. We established a mouse melanoma model for deleting Stat3 in melanocytes with specific expression of human hyperactive NRASQ61K in an Ink4a-deficient background, two frequent driver mutations in human melanoma. Mice devoid of Stat3 showed early disease onset with higher proliferation in primary tumors, but displayed significantly diminished lung, brain, and liver metastases. Whole-genome expression profiling of tumor-derived cells also showed a reduced invasion phenotype, which was further corroborated by 3D melanoma model analysis. Notably, loss or knockdown of STAT3 in mouse or human cells resulted in the upregulation of MITF and induction of cell proliferation. Mechanistically we show that STAT3-induced CAAT Box Enhancer Binding Protein (CEBP) expression was sufficient to suppress MITF transcription. Epigenetic analysis by ATAC-seq confirmed that CEBPa/b binding to the MITF enhancer region silenced the MITF locus. Finally, by classification of patient-derived melanoma samples, we show that STAT3 and MITF act antagonistically and hence contribute differentially to melanoma progression. We conclude that STAT3 is a driver of the metastatic process in melanoma and able to antagonize MITF via direct induction of CEBP family member transcription.

STAT5 is Expressed in CD34+/CD38- Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms.

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38- MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38- MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

High activation of STAT5A drives peripheral T-cell lymphoma and leukemia.

Recurrent gain-of-function mutations in the transcription factors STAT5A and much more in STAT5B were found in hematopoietic malignancies with the highest proportion in mature T- and natural killer-cell neoplasms (peripheral T-cell lymphoma, PTCL). No targeted therapy exists for these heterogeneous and often aggressive diseases. Given the shortage of models for PTCL, we mimicked graded STAT5A or STAT5B activity by expressing hyperactive Stat5a or STAT5B variants at low or high levels in the hematopoietic system of transgenic mice. Only mice with high activity levels developed a lethal disease resembling human PTCL. Neoplasia displayed massive expansion of CD8+ T cells and destructive organ infiltration. T cells were cytokine-hypersensitive with activated memory CD8+ T-lymphocyte characteristics. Histopathology and mRNA expression profiles revealed close correlation with distinct subtypes of PTCL. Pronounced STAT5 expression and activity in samples from patients with different subsets underline the relevance of JAK/STAT as a therapeutic target. JAK inhibitors or a selective STAT5 SH2 domain inhibitor induced cell death and ruxolitinib blocked T-cell neoplasia in vivo We conclude that enhanced STAT5A or STAT5B action both drive PTCL development, defining both STAT5 molecules as targets for therapeutic intervention.

Pre-clinical activity of combined LSD1 and mTORC1 inhibition in MLL-translocated acute myeloid leukaemia.

The histone demethylase lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML); tranylcypromine-derivative inhibitors induce loss of clonogenic activity and promote differentiation, in particular in the MLL-translocated molecular subtype of AML. In AML, the use of drugs in combination often delivers superior clinical activity. To identify genes and cellular pathways that collaborate with LSD1 to maintain the leukaemic phenotype, and which could be targeted by combination therapies, we performed a genome-wide CRISPR-Cas9 dropout screen. We identified multiple components of the amino acid sensing arm of mTORC1 signalling-RRAGA, MLST8, WDR24 and LAMTOR2-as cellular sensitizers to LSD1 inhibition. Knockdown of mTORC1 components, or mTORC1 pharmacologic inhibition, in combination with LSD1 inhibition enhanced differentiation in both cell line and primary cell settings, in vitro and in vivo, and substantially reduced the frequency of clonogenic primary human AML cells in a modelled minimal residual disease setting. Synergistic upregulation of a set of transcription factor genes associated with terminal monocytic lineage differentiation was observed. Thus, dual mTORC1 and LSD1 inhibition represents a candidate combination approach for enhanced differentiation in MLL-translocated AML which could be evaluated in early phase clinical trials.

Emerging Epigenetic Therapeutic Targets in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy for which treatment options have been largely limited to cytotoxic chemotherapy for the past four decades. Next-generation sequencing and other approaches have identified a spectrum of genomic and epigenomic alterations that contribute to AML initiation and maintenance. The key role of epigenetic modifiers and the reversibility of epigenetic changes have paved the way for evaluation of a new set of drug targets, and facilitated the design of novel candidate treatment strategies. More recently, seven new targeted therapies have been FDA-approved demonstrating successful implementation of the past decades' research. In this review, we will summarize the most recent advances in targeted therapeutics designed for a focused group of key epigenetic regulators in AML, outline their mechanism of action and their current status in clinical development. Furthermore, we will discuss promising new approaches for epigenetic targeted treatment in AML which are currently being tested in pre-clinical trials.

The ERBB-STAT3 Axis Drives Tasmanian Devil Facial Tumor Disease.

The marsupial Tasmanian devil (Sarcophilus harrisii) faces extinction due to transmissible devil facial tumor disease (DFTD). To unveil the molecular underpinnings of this transmissible cancer, we combined pharmacological screens with an integrated systems-biology characterization. Sensitivity to inhibitors of ERBB tyrosine kinases correlated with their overexpression. Proteomic and DNA methylation analyses revealed tumor-specific signatures linked to the evolutionary conserved oncogenic STAT3. ERBB inhibition blocked phosphorylation of STAT3 and arrested cancer cells. Pharmacological blockade of ERBB or STAT3 prevented tumor growth in xenograft models and restored MHC class I expression. This link between the hyperactive ERBB-STAT3 axis and major histocompatibility complex class I-mediated tumor immunosurveillance provides mechanistic insights into horizontal transmissibility and puts forward a dual chemo-immunotherapeutic strategy to save Tasmanian devils from DFTD. VIDEO ABSTRACT.

Implications of STAT3 and STAT5 signaling on gene regulation and chromatin remodeling in hematopoietic cancer.

STAT3 and STAT5 proteins are oncogenic downstream mediators of the JAK-STAT pathway. Deregulated STAT3 and STAT5 signaling promotes cancer cell proliferation and survival in conjunction with other core cancer pathways. Nuclear phosphorylated STAT3 and STAT5 regulate cell-type-specific transcription profiles via binding to promoter elements and exert more complex functions involving interaction with various transcriptional coactivators or corepressors and chromatin remodeling proteins. The JAK-STAT pathway can rapidly reshape the chromatin landscape upon cytokine, hormone, or growth factor stimulation and unphosphorylated STAT proteins also appear to be functional with respect to regulating chromatin accessibility. Notably, cancer genome landscape studies have implicated mutations in various epigenetic modifiers as well as the JAK-STAT pathway as underlying causes of many cancers, particularly acute leukemia and lymphomas. However, it is incompletely understood how mutations within these pathways can interact and synergize to promote cancer. We summarize the current knowledge of oncogenic STAT3 and STAT5 functions downstream of cytokine signaling and provide details on prerequisites for DNA binding and gene transcription. We also discuss key interactions of STAT3 and STAT5 with chromatin remodeling factors such as DNA methyltransferases, histone modifiers, cofactors, corepressors, and other transcription factors.

Pharmacologic inhibition of STAT5 in acute myeloid leukemia.

The transcription factor STAT5 is an essential downstream mediator of many tyrosine kinases (TKs), particularly in hematopoietic cancers. STAT5 is activated by FLT3-ITD, which is a constitutively active TK driving the pathogenesis of acute myeloid leukemia (AML). Since STAT5 is a critical mediator of diverse malignant properties of AML cells, direct targeting of STAT5 is of significant clinical value. Here, we describe the development and preclinical evaluation of a novel, potent STAT5 SH2 domain inhibitor, AC-4-130, which can efficiently block pathological levels of STAT5 activity in AML. AC-4-130 directly binds to STAT5 and disrupts STAT5 activation, dimerization, nuclear translocation, and STAT5-dependent gene transcription. Notably, AC-4-130 substantially impaired the proliferation and clonogenic growth of human AML cell lines and primary FLT3-ITD+ AML patient cells in vitro and in vivo. Furthermore, AC-4-130 synergistically increased the cytotoxicity of the JAK1/2 inhibitor Ruxolitinib and the p300/pCAF inhibitor Garcinol. Overall, the synergistic effects of AC-4-130 with TK inhibitors (TKIs) as well as emerging treatment strategies provide new therapeutic opportunities for leukemia and potentially other cancers.

The JAK2/STAT5 signaling pathway as a potential therapeutic target in canine mastocytoma.

BACKGROUND: Mastocytoma are frequently diagnosed cutaneous neoplasms in dogs. In non-resectable mastocytoma patients, novel targeted drugs are often applied. The transcription factor STAT5 has been implicated in the survival of human neoplastic mast cells (MC). Our study evaluated the JAK2/STAT5 pathway as a novel target in canine mastocytoma. MATERIALS AND METHODS: We employed inhibitors of JAK2 (R763, TG101348, AZD1480, ruxolitinib) and STAT5 (pimozide, piceatannol) and evaluated their effects on 2 mastocytoma cell lines, C2 and NI-1. RESULTS: Activated JAK2 and STAT5 were detected in both cell lines. The drugs applied were found to inhibit proliferation and survival in these cells with the following rank-order of potency: R763 > TG101348 > AZD1480 > pimozide > ruxolitinib > piceatannol. Moreover, synergistic anti-neoplastic effects were obtained by combining pimozide with KIT-targeting drugs (toceranib, masitinib, nilotinib, midostaurin) in NI-1 cells. CONCLUSION: The JAK2/STAT5 pathway is a novel potential target of therapy in canine mastocytoma.

Malignant Phenotypes in Metastatic Melanoma are Governed by SR-BI and its Association with Glycosylation and STAT5 Activation.

Metastatic melanoma is hallmarked by elevated glycolytic flux and alterations in cholesterol homeostasis. The contribution of cholesterol transporting receptors for the maintenance of a migratory and invasive phenotype is not well defined. Here, the scavenger receptor class B type I (SCARB1/SR-BI), a high-density lipoprotein (HDL) receptor, was identified as an estimator of melanoma progression in patients. We further aimed to identify the SR-BI-controlled gene expression signature and its related cellular phenotypes. On the basis of whole transcriptome analysis, it was found that SR-BI knockdown, but not functional inhibition of its cholesterol-transporting capacity, perturbed the metastasis-associated epithelial-to-mesenchymal transition (EMT) phenotype. Furthermore, SR-BI knockdown was accompanied by decreased migration and invasion of melanoma cells and reduced xenograft tumor growth. STAT5 is an important mediator of the EMT process and loss of SR-BI resulted in decreased glycosylation, reduced DNA binding, and target gene expression of STAT5. When human metastatic melanoma clinical specimens were analyzed for the abundance of SR-BI and STAT5 protein, a positive correlation was found. Finally, a novel SR-BI-regulated gene profile was determined, which discriminates metastatic from nonmetastatic melanoma specimens indicating that SR-BI drives gene expression contributing to growth at metastatic sites. Overall, these results demonstrate that SR-BI is a highly expressed receptor in human metastatic melanoma and is crucial for the maintenance of the metastatic phenotype.Implications: High SR-BI expression in melanoma is linked with increased cellular glycosylation and hence is essential for a metastasis-specific expression signature. Mol Cancer Res; 16(1); 135-46. ©2017 AACR.

Emerging therapeutic targets in myeloproliferative neoplasms and peripheral T-cell leukemia and lymphomas.

INTRODUCTION: Hematopoietic neoplasms are often driven by gain-of-function mutations of the JAK-STAT pathway together with mutations in chromatin remodeling and DNA damage control pathways. The interconnection between the JAK-STAT pathway, epigenetic regulation or DNA damage control is still poorly understood in cancer cell biology. Areas covered: Here, we focus on a broader description of mutational insights into myeloproliferative neoplasms and peripheral T-cell leukemia and lymphomas, since sequencing efforts have identified similar combinations of driver mutations in these diseases covering different lineages. We summarize how these pathways might be interconnected in normal or cancer cells, which have lost differentiation capacity and drive oncogene transcription. Expert opinion: Due to similarities in driver mutations including epigenetic enzymes, JAK-STAT pathway activation and mutated checkpoint control through TP53, we hypothesize that similar therapeutic approaches could be of benefit in these diseases. We give an overview of how driver mutations in these malignancies contribute to hematopoietic cancer initiation or progression, and how these pathways can be targeted with currently available tools.

Preloading with L-BPA, L-tyrosine and L-DOPA enhances the uptake of [18F]FBPA in human and mouse tumour cell lines.

Aim of this study was to investigate if cellular [18F]FBPA uptake can be increased upon preloading with amino acids. [18F]FBPA uptake was assessed in HuH-7, CaCo-2 and B16-F1 cells pretreated with different concentrations or incubation times of L-BPA, L-tyrosine or L-DOPA. Without preloading, highest uptake of [18F]FBPA was observed in B16-F1 cells, followed by CaCo-2 cells and HuH-7 cells. In all cell lines higher [18F]FBPA accumulation (up to 1.65-fold) was obtained with increasing L-BPA, L-DOPA and L-tyrosine concentrations.

Fibroblast growth factor receptor 4: a putative key driver for the aggressive phenotype of hepatocellular carcinoma.

Recently, we found upregulation of fibroblast growth factor receptor 4 (FGFR4) in a subset of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight into the role of FGFR4-mediated signalling for the aggressive behaviour of HCC cells. To overexpress FGFR4, hepatoma/hepatocarcinoma cells were transfected with a construct coding for FGFR4. For downmodulation of endogenous FGFR4, we used small interfering RNA or adenoviral infection with dominant-negative FGFR4 constructs being either kinase dead (kdFGFR4) or coding for the autoinhibitory soluble domain (solFGFR4). FGFR4 overexpression in non-tumourigenic hepatocarcinoma cells significantly reduced cell-matrix adhesion, enabled cells to grow anchorage-independently in soft agar, to disintegrate the lymph-/blood-endothelial barrier for intra-/extravasation of tumour cells and to form tumours in SCID mice. Transcriptome analysis revealed altered expression of genes involved in cell-matrix interactions. Conversely, in highly tumourigenic cell lines, kdFGFR4 or solFGFR4 lowered the proportion of cells in S phase of the cell cycle, enhanced the G0/G1 and G2/M-phase proportions, reduced anchorage-independent growth in vitro and attenuated disintegration of the lymph-/blood-endothelium and tumour formation in vivo. These findings were confirmed by altered expression profiles of genes being important for late stages of cell division. Deregulated FGFR4 expression appears to be one of the key drivers of the malignant phenotype of HCC cells. Accordingly, blockade of FGFR4-mediated signalling by soluble dominant-negative constructs, like solFGFR4, may be a feasible and promising therapeutic approach to antagonize aggressive behaviour of hepatoma/hepatocarcinoma cells.