Katja Pokrovskaja Tamm

Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies

The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR–Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.

Activation of STAT1 is required for interferon-alpha-mediated cell death.

Interferon-alpha (IFNα)-induced cell death of tumor cells is likely mediated through several signaling pathways. We previously demonstrated that blocking the activation of phosphoinositide-3-kinase, PI3K, or mammalian target of rapamycin, mTOR, partially inhibited apoptosis induced by IFNα. Here, we postulate using pharmacological inhibition and dominant negative mutants that activation of signal transducer and activator of transcription-1, STAT1, is also required for the cell death induced by IFNα. Inhibition of STAT1 tyrosine phosphorylation and DNA binding by a naturally occurring rotenoid deguelin also rescued U266 myeloma cell lines from IFNα-induced apoptosis. Deguelin had no effect on upstream Jak kinases or STAT2 phosphorylation suggesting the involvement of a yet unknown mechanism. Inhibition of STAT1 tyrosine phosphorylation and activity was independent of the known effects of deguelin on PI3K, Akt or mTOR as shown using selective pharmacological inhibitors against these kinases. The combination of deguelin and PI3K or mTOR antagonists further inhibited apoptosis suggesting that both the Jak-STAT and the PI3K/mTOR pathways contribute to the induction of apoptosis by IFNα in these cells. Over-expression of STAT1-Y701A or K410/413A mutants in Rhek-1 keratinocytes largely inhibited apoptosis further supporting the importance of STAT1 phosphorylation and activity for IFNα-induced cell death. Thus, at least two signaling pathways, one of which requires STAT1 activation, cooperate to mediate IFNα-induced apoptosis.

Cell crowding induces interferon regulatory factor 9, which confers resistance to chemotherapeutic drugs.

The mechanism of multicellular drug resistance, defined as the reduced efficacy of chemotherapeutic drugs in solid tumors is incompletely understood. Here we report that colon carcinoma cells cultured as 3D microtissues (spheroids) display dramatic increases in the expression of a subset of type I interferon‐(IFN)‐stimulated genes (ISGs). A similar gene signature was associated previously with resistance to radiation and chemotherapy, prompting us to examine the underlying biological mechanisms. Analysis of spheroids formed by different tumor cell lines and studies using knock‐down of gene expression showed that cell crowding leads to the induction of IFN regulatory factor‐9 (IRF9) which together with STAT2 and independently of IFNs, is necessary for ISG upregulation. Increased expression of IRF9 alone was sufficient to induce the ISG subset in monolayer cells and to confer increased resistance to clinically used cytotoxic drugs. Our data reveal a novel mechanism of regulation of a subset of ISGs, leading to drug resistance in solid tumors.

Patients with activated B-cell like diffuse large B-cell lymphoma in high and low infectious disease areas have different inflammatory gene signatures.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with an association with inflammation and viral infections. We hypothesize that environmental factors may be involved in the pathogenesis of DLBCL. In this study, we compared gene expression profiles of lymph node tissues from patients with DLBCL from two different geographical areas with diverse environmental exposures. Specimens from Egyptian and Swedish patients with DLBCL as well as controls were studied. Gene expression analysis using microarray and quantitative polymerase chain reaction demonstrated significantly higher expression of signal transducer and activator of transcription 3 (STAT3) in Swedish as compared to Egyptian patients and control materials from both countries. This was confirmed at protein level using confocal microscopy. The receptor tyrosine kinase ROR1, a “survival factor” for malignant cells, was overexpressed and significantly related to the STAT3 expression pattern. The difference in the expression of genes involved in inflammatory responses and in the tumorigenic process of DLBCL might relate to infectious agents and/or other environmental exposures.

Identification of novel small molecules that inhibit STAT3-dependent transcription and function

Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z’ = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.

An antisense RNA capable of modulating the expression of the tumor suppressor microRNA-34a

The microRNA-34a is a well-studied tumor suppressor microRNA (miRNA) and a direct downstream target of TP53 with roles in several pathways associated with oncogenesis, such as proliferation, cellular growth, and differentiation. Due to its broad tumor suppressive activity, it is not surprising that miR34a expression is altered in a wide variety of solid tumors and hematological malignancies. However, the mechanisms by which miR34a is regulated in these cancers is largely unknown. In this study, we find that a long noncoding RNA transcribed antisense to the miR34a host gene, is critical for miR34a expression and mediation of its cellular functions in multiple types of human cancer. We name this long noncoding RNA lncTAM34a, and characterize its ability to facilitate miR34a expression under different types of cellular stress in both TP53-deficient and wild-type settings.

Targeting autophagy by small molecule inhibitors of vacuolar protein sorting 34 (Vps34) improves the sensitivity of breast cancer cells to Sunitinib

Resistance to chemotherapy is a challenging problem for treatment of cancer patients and autophagy has been shown to mediate development of resistance. In this study we systematically screened a library of 306 known anti-cancer drugs for their ability to induce autophagy using a cell-based assay. 114 of the drugs were classified as autophagy inducers; for 16 drugs, the cytotoxicity was potentiated by siRNA-mediated knock-down of Atg7 and Vps34. These drugs were further evaluated in breast cancer cell lines for autophagy induction, and two tyrosine kinase inhibitors, Sunitinib and Erlotinib, were selected for further studies. For the pharmacological inhibition of autophagy, we have characterized here a novel highly potent selective inhibitor of Vps34, SB02024. SB02024 blocked autophagy in vitro and reduced xenograft growth of two breast cancer cell lines, MDA-MB-231 and MCF-7, in vivo. Vps34 inhibitor significantly potentiated cytotoxicity of Sunitinib and Erlotinib in MCF-7 and MDA-MB-231 in vitro in monolayer cultures and when grown as multicellular spheroids. Our data suggests that inhibition of autophagy significantly improves sensitivity to Sunitinib and Erlotinib and that Vps34 is a promising therapeutic target for combination strategies in breast cancer.