Myeloid Disease Mutations of Splicing Factor SRSF2 Cause G2-M Arrest and Skewed Differentiation of Human Hematopoietic Stem and Progenitor Cells.

Myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia, are characterized by abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). Reports on analysis of bone marrow samples from patients have revealed a high incidence of mutations in splicing factors in early stem and progenitor cell clones, but the mechanisms underlying transformation of HSPCs harboring these mutations remain unknown. Using ex vivo cultures of primary human CD34+ cells as a model, we find that mutations in splicing factors SRSF2 and U2AF1 exert distinct effects on proliferation and differentiation of HSPCs. SRSF2 mutations cause a dramatic inhibition of proliferation via a G2-M phase arrest and induction of apoptosis. U2AF1 mutations, conversely, do not significantly affect proliferation. Mutations in both SRSF2 and U2AF1 cause abnormal differentiation by skewing granulo-monocytic differentiation toward monocytes but elicit diverse effects on megakaryo-erythroid differentiation. The SRSF2 mutations skew differentiation toward megakaryocytes whereas U2AF1 mutations cause an increase in the erythroid cell populations. These distinct functional consequences indicate that SRSF2 and U2AF1 mutations have cell context-specific effects and that the generation of myeloid disease phenotype by mutations in the genes coding these two proteins likely involves different intracellular mechanisms. Stem Cells 2018;36:1663-1675.

Defining and targeting macrophage heterogeneity in the mammary gland and breast cancer.

INTRODUCTION: Macrophages are innate immune cells that are associated with extensive phenotypic and functional plasticity and contribute to normal development, tissue homeostasis, and diseases such as cancer. In this review, we discuss the heterogeneity of tissue resident macrophages in the normal mammary gland and tumor-associated macrophages in breast cancer. Tissue resident macrophages are required for mammary gland development, where they have been implicated in promoting extracellular matrix remodeling, apoptotic clearance, and cellular crosstalk. In the context of cancer, tumor-associated macrophages are key drivers of growth and metastasis via their ability to promote matrix remodeling, angiogenesis, lymphangiogenesis, and immunosuppression. METHOD: We identified and summarized studies in Pubmed that describe the phenotypic and functional heterogeneity of macrophages and the implications of targeting individual subsets, specifically in the context of mammary gland development and breast cancer. We also identified and summarized recent studies using single-cell RNA sequencing to identify and describe macrophage subsets in human breast cancer samples. RESULTS: Advances in single-cell RNA sequencing technologies have yielded nuances in macrophage heterogeneity, with numerous macrophage subsets identified in both the normal mammary gland and breast cancer tissue. Macrophage subsets contribute to mammary gland development and breast cancer progression in differing ways, and emerging studies highlight a role for spatial localization in modulating their phenotype and function. CONCLUSION: Understanding macrophage heterogeneity and the unique functions of each subset in both normal mammary gland development and breast cancer progression may lead to more promising targets for the treatment of breast cancer.

Targeting the NF-κB pathway enhances responsiveness of mammary tumors to JAK inhibitors.

Interactions between tumor cells and the tumor microenvironment are critical for tumor growth, progression, and response to therapy. Effective targeting of oncogenic signaling pathways in tumors requires an understanding of how these therapies impact both tumor cells and cells within the tumor microenvironment. One such pathway is the janus kinase (JAK)/signal transducer and activator or transcription (STAT) pathway, which is activated in both breast cancer cells and in tumor associated macrophages. This study demonstrates that exposure of macrophages to JAK inhibitors leads to activation of NF-κB signaling, which results in increased expression of genes known to be associated with therapeutic resistance. Furthermore, inhibition of the NF-κB pathway improves the ability of ruxolitinib to reduce mammary tumor growth in vivo. Thus, the impact of the tumor microenvironment is an important consideration in studying breast cancer and understanding such mechanisms of resistance is critical to development of effective targeted therapies.

Phosphatase PHLPP2 regulates the cellular response to metabolic stress through AMPK.

PHLPP2 is a member of the PHLPP family of phosphatases, known to suppress cell growth by inhibiting proliferation or promoting apoptosis. Oncogenic kinases Akt, S6K, and PKC, and pro-apoptotic kinase Mst1, have been recognized as functional targets of the PHLPP family. However, we observed that, in T-leukemia cells subjected to metabolic stress from glucose limitation, PHLPP2 specifically targets the energy-sensing AMP-activated protein kinase, pAMPK, rather than Akt or S6K. PHLPP2 dephosphorylates pAMPK in several other human cancer cells as well. PHLPP2 and pAMPK interact with each other, and the pleckstrin homology (PH) domain on PHLPP2 is required for their interaction, for dephosphorylating and inactivating AMPK, and for the apoptotic response of the leukemia cells to glucose limitation. Silencing PHLPP2 protein expression prolongs the survival of leukemia cells subjected to severe glucose limitation by promoting a switch to AMPK-mediated fatty acid oxidation for energy generation. Thus, this study reveals a novel role for PHLPP2 in suppressing a survival response mediated through AMPK signaling. Given the multiple ways in which PHLPP phosphatases act to oppose survival signaling in cancers and the pivotal role played by AMPK in redox homeostasis via glucose and fatty acid metabolism, the revelation that AMPK is a target of PHLPP2 could lead to better therapeutics directed both at cancer and at metabolic diseases.

Hypoxia promotes erythroid differentiation through the development of progenitors and proerythroblasts.

Oxygen is a critical noncellular component of the bone marrow microenvironment that plays an important role in the development of hematopoietic cell lineages. In this study, we investigated the impact of low oxygen (hypoxia) on ex vivo myeloerythroid differentiation of human cord blood-derived CD34+ hematopoietic stem and progenitor cells. We characterized the culture conditions to demonstrate that low oxygen inhibits cell proliferation and causes a metabolic shift in the stem and progenitor populations. We found that hypoxia promotes erythroid differentiation by supporting the development of progenitor populations. Hypoxia also increases the megakaryoerythroid potential of the common myeloid progenitors and the erythroid potential of megakaryoerythroid progenitors and significantly accelerates maturation of erythroid cells. Specifically, we determined that hypoxia promotes the loss of CD71 and the appearance of the erythroid markers CD235a and CD239. Further, evaluation of erythroid populations revealed a hypoxia-induced increase in proerythroblasts and in enucleation of CD235a+ cells. These results reveal the extensive role of hypoxia at multiple steps during erythroid development. Overall, our work establishes a valuable model for further investigations into the relationship between erythroid progenitors and/or erythroblast populations and their hypoxic microenvironment.

Novel small-molecule inhibitor of apurinic/apyrimidinic endonuclease 1 blocks proliferation and reduces viability of glioblastoma cells.

Apurinic/apyrimidinic (AP) endonuclease 1 (Ape1) is an essential DNA repair protein that plays a critical role in repair of AP sites via base excision repair. Ape1 has received attention as a druggable oncotherapeutic target, especially for treating intractable cancers such as glioblastoma. The goal of this study was to identify small-molecule inhibitors of Ape1 AP endonuclease. For this purpose, a fluorescence-based high-throughput assay was used to screen a library of 60,000 small-molecule compounds for ability to inhibit Ape1 AP endonuclease activity. Four compounds with IC(50) values less than 10 microM were identified, validated, and characterized. One of the most promising compounds, designated Ape1 repair inhibitor 03 [2,4,9-trimethylbenzo[b][1,8]-naphthyridin-5-amine; AR03), inhibited cleavage of AP sites in vivo in SF767 glioblastoma cells and in vitro in whole cell extracts and inhibited purified human Ape1 in vitro. AR03 has low affinity for double-stranded DNA and weakly inhibits the Escherichia coli endonuclease IV, requiring a 20-fold higher concentration than for inhibition of Ape1. AR03 also potentiates the cytotoxicity of methyl methanesulfonate and temozolomide in SF767 cells. AR03 is chemically distinct from the previously reported small-molecule inhibitors of Ape1. AR03 is a novel small-molecule inhibitor of Ape1, which may have potential as an oncotherapeutic drug for treating glioblastoma and other cancers.

Semi-automated high-throughput fluorescent intercalator displacement-based discovery of cytotoxic DNA binding agents from a large compound library.

High-throughput fluorescent intercalator displacement (HT-FID) was adapted to the semi-automated screening of a commercial compound library containing 60,000 molecules resulting in the discovery of cytotoxic DNA-targeted agents. Although commercial libraries are routinely screened in drug discovery efforts, the DNA binding potential of the compounds they contain has largely been overlooked. HT-FID led to the rapid identification of a number of compounds for which DNA binding properties were validated through demonstration of concentration-dependent DNA binding and increased thermal melting of A/T- or G/C-rich DNA sequences. Selected compounds were assayed further for cell proliferation inhibition in glioblastoma cells. Seven distinct compounds emerged from this screening procedure that represent structures unknown previously to be capable of targeting DNA leading to cell death. These agents may represent structures worthy of further modification to optimally explore their potential as cytotoxic anti-cancer agents. In addition, the general screening strategy described may find broader impact toward the rapid discovery of DNA targeted agents with biological activity.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells.

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Blocking Nerve Growth Factor Signaling Reduces the Neural Invasion Potential of Pancreatic Cancer Cells.

Perineural invasion (PNI) is thought to be one of the factors responsible for the high rate of tumor recurrence after surgery and the pain generation associated with pancreatic cancer. Signaling via the nerve growth factor (NGF) pathway between pancreatic cancer cells and the surrounding nerves has been implicated in PNI, and increased levels of these proteins have been correlated to poor prognosis. In this study, we examine the molecular mechanism of the NGF signaling pathway in PNI in pancreatic cancer. We show that knocking down NGF or its receptors, TRKA and p75NTR, or treatment with GW441756, a TRKA kinase inhibitor, reduces the proliferation and migration of pancreatic cancer cells in vitro. Furthermore, pancreatic cancer cells migrate towards dorsal root ganglia (DRG) in a co-culture assay, indicating a paracrine NGF signaling between the DRGs and pancreatic cancer cells. Knocking down the expression of NGF pathway proteins or inhibiting the activity of TRKA by GW441756 reduced the migratory ability of Mia PaCa2 towards the DRGs. Finally, blocking NGF signaling by NGF neutralizing antibodies or GW441756 inhibited the neurite formation in PC-12 cells in response to conditioned media from pancreatic cancer cells, indicating a reciprocal signaling pathway between the pancreatic cancer cells and nerves. Our results indicate that NGF signaling pathway provides a potential target for developing molecularly targeted therapies to decrease PNI and reduce pain generation. Since there are several TRKA antagonists currently in early clinical trials they could now be tested in the clinical situation of pancreatic cancer induced pain.

Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5 year survival rate of 20-30 %. Various factors have been implicated in the pathogenesis of ACC including dysregulation of the G2/M transition and aberrant activity of p53 and MDM2. Polo-like kinase 1 (PLK-1) negatively modulates p53 functioning, promotes MDM2 activity through its phosphorylation, and is involved in the G2/M transition. Gene expression profiling of 44 ACC samples showed that increased expression of PLK-1 in 29 % of ACC. Consequently, we examined PLK-1's role in the modulation of the p53 signaling pathway in adrenocortical cancer. METHODS: We used siRNA knock down PLK-1 and pharmacological inhibition of PLK-1 and MDM2 ACC cell lines SW-13 and H295R. We examined viability, protein expression, p53 transactivation, and induction of apoptosis. RESULTS: Knocking down expression of PLK-1 with siRNA or inhibition of PLK-1 by a small molecule inhibitor, BI-2536, resulted in a loss of viability of up to 70 % in the ACC cell lines H295R and SW-13. In xenograft models, BI-2536 demonstrated marked inhibition of growth of SW-13 with less inhibition of H295R. BI-2536 treatment resulted in a decrease in mutant p53 protein in SW-13 cells but had no effect on wild-type p53 protein levels in H295R cells. Additionally, inhibition of PLK-1 restored wild-type p53's transactivation and apoptotic functions in H295R cells, while these functions of mutant p53 were restored only to a smaller extent. Furthermore, inhibition of MDM2 with nutlin-3 reduced the viability of both the ACC cells and also reactivated wild-type p53's apoptotic function. Inhibition of PLK-1 sensitized the ACC cell lines to MDM2 inhibition and this dual inhibition resulted in an additive apoptotic response in H295R cells with wild-type p53. CONCLUSIONS: These preclinical studies suggest that targeting p53 through PLK-1 is an attractive chemotherapy strategy warranting further investigation in adrenocortical cancer.

Perineural invasion and associated pain in pancreatic cancer.

Perineural invasion (PNI) is a prominent characteristic of pancreatic cancer. PNI is a process whereby cancer cells invade the surrounding nerves, thus providing an alternative route for metastatic spread and pain generation. PNI is thought to be an indicator of aggressive tumour behaviour and has been shown to correlate with poor prognosis of patients with pancreatic cancer. Recent studies demonstrated that some signalling molecules and pathways that are involved in PNI are also involved in pain generation. Targeting these signalling pathways has shown some promise in alleviating pain and reducing PNI, which could potentially improve treatment outcomes for patients with pancreatic cancer.

Going ape as an approach to cancer therapeutics.

The DNA base excision repair (BER) pathway repairs alkylation and oxidative DNA damage caused by endogenous and exogenous agents, including chemotherapeutic agents. Upon removal of the damaged base AP endonuclease 1 (Ape1), a critical component of the pathway cleaves the abasic site to facilitate repair. Ape1 is a multifunctional protein which plays a role not only in DNA repair but it also functions as a reduction-oxidation factor, known as Ref-1 in the literature, to increase the DNA binding ability of several transcription factors involved in different growth signaling pathways. Elevated levels of Ape1 have been linked to resistance to chemotherapy, poor prognosis, and poor survival. Reducing the amount of Ape1 protein in cancer cells and tumors using RNA interference and anti-sense oligonucleotide technology sensitizes mammalian tumor cells to a variety of laboratory and chemotherapeutic agents. Therefore, selective inhibition of Ape1's DNA repair activity is a promising avenue to develop novel cancer therapeutics.