Schlafens: Emerging Therapeutic Targets.

The interferon (IFN) family of immunomodulatory cytokines has been a focus of cancer research for over 50 years with direct and indirect implications in cancer therapy due to their properties to inhibit malignant cell proliferation and modulate immune responses. Among the transcriptional targets of the IFNs is a family of genes referred to as Schlafens. The products of these genes, Schlafen proteins, exert important roles in modulating cellular proliferation, differentiation, immune responses, viral replication, and chemosensitivity of malignant cells. Studies have demonstrated that abnormal expression of various Schlafens contributes to the pathophysiology of various cancers. Schlafens are now emerging as promising biomarkers and potentially attractive targets for drug development in cancer research. Here, we highlight research suggesting the use of Schlafens as cancer biomarkers and the rationale for the development of specific drugs targeting Schlafen proteins.

Targeting CHAF1B Enhances IFN Activity against Myeloproliferative Neoplasm Cells.

Interferons (IFNs) are cytokines with potent antineoplastic and antiviral properties. IFNα has significant clinical activity in the treatment of myeloproliferative neoplasms (MPN), but the precise mechanisms by which it acts are not well understood. Here, we demonstrate that chromatin assembly factor 1 subunit B (CHAF1B), an Unc-51-like kinase 1 (ULK1)-interactive protein in the nuclear compartment of malignant cells, is overexpressed in patients with MPN. Remarkably, targeted silencing of CHAF1B enhances transcription of IFNα-stimulated genes and promotes IFNα-dependent antineoplastic responses in primary MPN progenitor cells. Taken together, our findings indicate that CHAF1B is a promising newly identified therapeutic target in MPN and that CHAF1B inhibition in combination with IFNα therapy might offer a novel strategy for treating patients with MPN. Significance: Our findings raise the potential for clinical development of drugs targeting CHAF1B to enhance IFN antitumor responses in the treatment of patients with MPN and should have important clinical translational implications for the treatment of MPN and possibly in other malignancies.

Prolylcarboxypeptidase promotes IGF1R/HER3 signaling and is a potential target to improve endocrine therapy response in estrogen receptor positive breast cancer.

Prolylcarboxypeptidase (PRCP) is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. Previous studies have linked PRCP to blood-pressure and appetite control through its ability to cleave peptide substrates such as angiotensin II and α-MSH. A potential role for PRCP in cancer has to date not been widely appreciated. Endocrine therapy resistance in breast cancer is an enduring clinical problem mediated in part by aberrant receptor tyrosine kinase (RTK) signaling. We previously found PRCP overexpression promoted 4-hydroxytamoxifen (4-OHT) resistance in estrogen receptor-positive (ER+) breast cancer cells. Currently, we tested the potential association between PRCP with breast cancer patient outcome and RTK signaling, and tumor responsiveness to endocrine therapy. We found high PRCP protein levels in ER+ breast tumors associates with worse outcome and earlier recurrence in breast cancer patients, including patients treated with TAM. We found a PRCP specific inhibitor (PRCPi) enhanced the response of ER+ PDX tumors and MCF7 tumors to endoxifen, an active metabolite of TAM in mice. We found PRCP increased IGF1R/HER3 signaling and AKT activation in ER+ breast cancer cells that was blocked by PRCPi. Thus, PRCP is an adverse prognostic marker in breast cancer and a potential target to improve endocrine therapy in ER+ breast cancers.

SLFN11 negatively regulates non-canonical NFkB signaling to promote glioblastoma progression.

Glioblastoma (GBM) is an aggressive and incurable brain tumor in nearly all instances, whose disease progression is driven in part by the glioma stem cell (GSC) subpopulation. Here, we explored the effects of Schlafen family member 11 (SLFN11) in the molecular, cellular and tumor biology of GBM. CRISPR/Cas9 mediated knockout (KO) of SLFN11 inhibited GBM cell proliferation and neurosphere growth and was associated with reduced expression of progenitor/stem cell marker genes, such as NES, SOX2 and CD44. Loss of SLFN11 stimulated expression of NF-κB target genes, consistent with a negative regulatory role for SLFN11 on the NF-κB pathway. Further, our studies identify p21 as a direct transcriptional target of NF-κB2 in GBM whose expression was stimulated by loss of SLFN11. Genetic disruption of SLFN11 blocked GBM growth and significantly extended survival in an orthotopic patient-derived xenograft model. Together, our results identify SLFN11 as a novel component of signaling pathways that contribute to GBM and GSC with implications for future diagnostic and therapeutic strategies.

Regulation of IFNα-induced expression of the short ACE2 isoform by ULK1.

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been shown to hijack angiotensin converting enzyme 2 (ACE2) for entry into mammalian cells. A short isoform of ACE2, termed deltaACE2 (dACE2), has recently been identified. In contrast to ACE2, the short dACE2 isoform lacks the ability to bind the spike protein of SARS-CoV-2. Several studies have proposed that expression of ACE2 and/or dACE2 is induced by interferons (IFNs). Here, we report that drug-targeted inhibition or silencing of Unc51-like kinase 1 (ULK1) results in repression of type I IFN-induced expression of the dACE2 isoform. Notably, dACE2 is expressed in various squamous tumors. In efforts to identify pharmacological agents that target this pathway, we found that fisetin, a natural flavonoid, is an ULK1 inhibitor that decreases type I IFN-induced dACE2 expression. Taken together, our results establish a requirement for ULK1 in the regulation of type I IFN-induced transcription of dACE2 and raise the possibility of clinical translational applications of fisetin as a novel ULK1 inhibitor.

Prolyl Carboxypeptidase Maintains Receptor Tyrosine Kinase Signaling and Is a Potential Therapeutic Target in Triple Negative Breast Cancer.

TNBC is an aggressive cancer sub-type with limited treatment options and poor prognosis. New therapeutic targets are needed to improve outcomes in TNBC patients. PRCP is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. A role for PRCP in TNBC or other cancers, and its potential as a therapy target has not yet been tested. In the current study, we found high tumor expression of PRCP associates with worse outcome and earlier recurrence in TNBC patients. Knockdown of PRCP or treatment with a small molecule PRCP inhibitor blocked proliferation and survival in TNBC cell lines and inhibited growth of TNBC tumors in mice. Mechanistically, we found PRCP maintains signaling from multiple receptor tyrosine kinases (RTKs), potentially by promoting crosstalk between RTKs and G-protein coupled receptors (GPCRs). Lastly, we found that the PRCP inhibitor caused synergistic killing of TNBC cells when combined with the EGFR and ErbB2 inhibitor lapatinib. Our results suggest that PRCP is potential prognostic marker for TNBC patient outcome and a novel therapeutic target for TNBC treatment.

Inhibitors of Jumonji C domain-containing histone lysine demethylases overcome cisplatin and paclitaxel resistance in non-small cell lung cancer through APC/Cdh1-dependent degradation of CtIP and PAF15.

The Jumonji C domain-containing family of histone lysine demethylases (Jumonji KDMs) have emerged as promising cancer therapy targets. These enzymes remove methyl groups from various histone lysines and, in turn, regulate processes including chromatin compaction, gene transcription, and DNA repair. Small molecule inhibitors of Jumonji KDMs have shown promise in preclinical studies against non-small cell lung cancer (NSCLC) and other cancers. However, how these inhibitors influence cancer therapy responses and/or DNA repair is incompletely understood. In this study, we established cell line and PDX tumor model systems of cisplatin and paclitaxel-resistant NSCLC. We showed that resistant cells and tumors express high levels of Jumonji-KDMs. Knockdown of individual KDMs or treatment with a pan-Jumonji KDM inhibitor sensitized the cells and tumors to cisplatin and paclitaxel and blocked NSCLC in vivo tumor growth. Mechanistically, we found inhibition of Jumonji-KDMs triggers APC/Cdh1-dependent degradation of CtIP and PAF15, two DNA repair proteins that promote repair of cisplatin and paclitaxel-induced DNA lesions. Knockdown of CtIP and PAF15 sensitized resistant cells to cisplatin, indicating their degradation when Jumonji KDMs are inhibited contributes to cisplatin sensitivity. Our results support the idea that Jumonji-KDMs are a targetable barrier to effective therapy responses in NSCLC. Inhibition of Jumonji KDMs increases therapy (cisplatin/paclitaxel) sensitivity in NSCLC cells, at least in part, by promoting APC/Cdh1-dependent degradation of CtIP and PAF15.

RBL2/DREAM-mediated repression of the Aurora kinase A/B pathway determines therapy responsiveness and outcome in p53 WT NSCLC.

Wild-type p53 is a stress-responsive transcription factor and potent tumor suppressor. P53 activates or represses genes involved in cell cycle progression or apoptosis in order to arrest the cell cycle or induce cell death. Transcription repression by p53 is indirect and requires repressive members of the RB-family (RB1, RBL1, RBL2) and formation of repressor complexes of RB1-E2F and RBL1/RBL2-DREAM. Many aurora kinase A/B (AURKA/B) pathway genes are repressed in a p53-DREAM-dependent manner. We found heightened expression of RBL2 and reduced expression of AURKA/B pathway genes is associated with improved outcomes in p53 wild-type but not p53 mutant non-small cell lung cancer (NSCLC) patients. Knockdown of p53, RBL2, or the DREAM component LIN37 increased AURKA/B pathway gene expression and reduced paclitaxel and radiation toxicity in NSCLC cells. In contrast, pharmacologic inhibition of AURKA/B or knockdown of AURKA/B pathway components increased paclitaxel and IR sensitivity. The results support a model in which p53-RBL2-DREAM-mediated repression of the AURKA/B pathway contributes to tumor suppression, improved tumor therapy responses, and better outcomes in p53 wild-type NSCLCs.

Schlafen 5 as a novel therapeutic target in pancreatic ductal adenocarcinoma.

We provide evidence that a member of the human Schlafen (SLFN) family of proteins, SLFN5, is overexpressed in human pancreatic ductal adenocarcinoma (PDAC). Targeted deletion of SLFN5 results in decreased PDAC cell proliferation and suppresses PDAC tumorigenesis in in vivo PDAC models. Importantly, high expression levels of SLFN5 correlate with worse outcomes in PDAC patients, implicating SLFN5 in the pathophysiology of PDAC that leads to poor outcomes. Our studies establish novel regulatory effects of SLFN5 on cell cycle progression through binding/blocking of the transcriptional repressor E2F7, promoting transcription of key genes that stimulate S phase progression. Together, our studies suggest an essential role for SLFN5 in PDAC and support the potential for developing new therapeutic approaches for the treatment of pancreatic cancer through SLFN5 targeting.

Fatty acid oxidation and autophagy promote endoxifen resistance and counter the effect of AKT inhibition in ER-positive breast cancer cells.

Tamoxifen (TAM) is the first-line endocrine therapy for estrogen receptor-positive (ER+) breast cancer (BC). However, acquired resistance occurs in ∼50% cases. Meanwhile, although the PI3K/AKT/mTOR pathway is a viable target for treatment of endocrine therapy-refractory patients, complex signaling feedback loops exist, which can counter the effectiveness of inhibitors of this pathway. Here, we analyzed signaling pathways and metabolism in ER+ MCF7 BC cell line and their TAM-resistant derivatives that are co-resistant to endoxifen using immunoblotting, quantitative polymerase chain reaction, and the Agilent Seahorse XF Analyzer. We found that activation of AKT and the energy-sensing kinase AMPK was increased in TAM and endoxifen-resistant cells. Furthermore, ERRα/PGC-1ß and their target genes MCAD and CPT-1 were increased and regulated by AMPK, which coincided with increased fatty acid oxidation (FAO) and autophagy in TAM-resistant cells. Inhibition of AKT feedback-activates AMPK and ERRα/PGC-1ß-MCAD/CPT-1 with a consequent increase in FAO and autophagy that counters the therapeutic effect of endoxifen and AKT inhibitors. Therefore, our results indicate increased activation of AKT and AMPK with metabolic reprogramming and increased autophagy in TAM-resistant cells. Simultaneous inhibition of AKT and FAO/autophagy is necessary to fully sensitize resistant cells to endoxifen.

Combined PI3Kα-mTOR Targeting of Glioma Stem Cells.

Glioblastoma (GBM) is the most common and lethal primary intrinsic tumour of the adult brain and evidence indicates disease progression is driven by glioma stem cells (GSCs). Extensive advances in the molecular characterization of GBM allowed classification into proneural, mesenchymal and classical subtypes, and have raised expectations these insights may predict response to targeted therapies. We utilized GBM neurospheres that display GSC characteristics and found activation of the PI3K/AKT pathway in sphere-forming cells. The PI3Kα selective inhibitor alpelisib blocked PI3K/AKT activation and inhibited spheroid growth, suggesting an essential role for the PI3Kα catalytic isoform. p110α expression was highest in the proneural subtype and this was associated with increased phosphorylation of AKT. Further, employing the GBM BioDP, we found co-expression of PIK3CA with the neuronal stem/progenitor marker NES was associated with poor prognosis in PN GBM patients, indicating a unique role for PI3Kα in PN GSCs. Alpelisib inhibited GSC neurosphere growth and these effects were more pronounced in GSCs of the PN subtype. The antineoplastic effects of alpelisib were substantially enhanced when combined with pharmacologic mTOR inhibition. These findings identify the alpha catalytic PI3K isoform as a unique therapeutic target in proneural GBM and suggest that pharmacological mTOR inhibition may sensitize GSCs to selective PI3Kα inhibition.

Type I Interferon (IFN)-Regulated Activation of Canonical and Non-Canonical Signaling Pathways.

For several decades there has been accumulating evidence implicating type I interferons (IFNs) as key elements of the immune response. Therapeutic approaches incorporating different recombinant type I IFN proteins have been successfully employed to treat a diverse group of diseases with significant and positive outcomes. The biological activities of type I IFNs are consequences of signaling events occurring in the cytoplasm and nucleus of cells. Biochemical events involving JAK/STAT proteins that control transcriptional activation of IFN-stimulated genes (ISGs) were the first to be identified and are referred to as "canonical" signaling. Subsequent identification of JAK/STAT-independent signaling pathways, critical for ISG transcription and/or mRNA translation, are denoted as "non-canonical" or "non-classical" pathways. In this review, we summarize these signaling cascades and discuss recent developments in the field, specifically as they relate to the biological and clinical implications of engagement of both canonical and non-canonical pathways.

Prolyl endopeptidase inhibitor Y-29794 blocks the IRS1-AKT-mTORC1 pathway and inhibits survival and in vivo tumor growth of triple-negative breast cancer.

Prolyl endopeptidase (PREP), also known as prolyl oligopeptidase (POP), is an enzyme that cleaves short peptides (<30 amino acids in length) on the C-terminal side of proline. PREP is highly expressed in multiple carcinomas and is a potential target for cancer therapy. A potent inhibitor of PREP, Y-29794, causes long-lasting inhibition of PREP in mouse tissues. However, there are no reports on Y-29794 effects on cancer cell and tumor proliferation. Using cell line models of aggressive triple-negative breast cancer (TNBC), we show here that Y-29794 inhibited proliferation and induced death in multiple TNBC cell lines. Cell death induced by Y-29794 coincided with inhibition of the IRS1-AKT-mTORC1 survival signaling pathway, although stable depletion of PREP alone was not sufficient to reduce IRS1-AKT-mTORC1 signaling or induce death. These results suggest that Y-29794 elicits its cancer cell killing effect by targeting other mechanisms in addition to PREP. Importantly, Y-29794 inhibited tumor growth when tested in xenograft models of TNBC in mice. Induction of cell death in culture and inhibition of xenograft tumor growth support the potential utility of Y-29794 or its derivatives as a treatment option for TNBC tumors.

The histone demethylase JMJD2B is critical for p53-mediated autophagy and survival in Nutlin-treated cancer cells.

Autophagy promotes cancer cell survival in response to p53 activation by the anticancer agent Nutlin-3a (Nutlin). We reported previously that Nutlin kills MDM2-amplified cancer cells and that this killing is associated with an inhibition of glucose metabolism, reduced α-ketoglutarate (α-KG) levels, and reduced autophagy. In the current report, using SJSA1, U2OS, A549, and MHM cells, we found that Nutlin alters histone methylation in an MDM2 proto-oncogene-dependent manner and that this, in turn, regulates autophagy-related gene (ATG) expression and cell death. In MDM2-amplified cells, Nutlin increased histone (H) 3 lysine (K) 9 and K36 trimethylation (me3) coincident with reduced autophagy and increased apoptosis. Blocking histone methylation restored autophagy and rescued these cells from Nutlin-induced killing. In MDM2-nonamplified cells, H3K9me3 and H3K36me3 levels were either reduced or not changed by the Nutlin treatment, and this coincided with increased autophagy and cell survival. Blocking histone demethylation reduced autophagy and sensitized these cells to Nutlin-induced killing. Further experiments suggested that MDM2 amplification increases histone methylation in Nutlin-treated cells by causing depletion of the histone demethylase Jumonji domain-containing protein 2B (JMJD2B). Finally, JMJD2B knockdown or inhibition increased H3K9/K36me3 levels, decreased ATG gene expression and autophagy, and sensitized MDM2-nonamplified cells to apoptosis. Together, these results support a model in which MDM2- and JMJD2B-regulated histone methylation levels modulate ATG gene expression, autophagy, and cell fate in response to the MDM2 antagonist Nutlin-3a.

JMJD2 promotes acquired cisplatin resistance in non-small cell lung carcinoma cells.

Platinum-based drugs such as cisplatin (CP) are the first-line chemotherapy for non-small-cell lung carcinoma (NSCLC). Unfortunately, NSCLC has a low response rate to CP and acquired resistance always occurs. Histone methylation regulates chromatin structure and is implicated in DNA repair. We hypothesize histone methylation regulators are involved in CP resistance. We therefore screened gene expression of known histone methyltransferases and demethylases in three NSCLC cell lines with or without acquired resistance to CP. JMJD2s are a family of histone demethylases that remove tri-methyl groups from H3K9 and H3K36. We found expression of several JMJD2 family genes upregulated in CP-resistant cells, with JMJD2B expression being upregulated in all three CP-resistant NSCLC cell lines. Further analysis showed increased JMJD2 protein expression coincided with decreased H3K9me3 and H3K36me3. Chemical inhibitors of JMJD2-family proteins increased H3K9me3 and H3K36me3 levels and sensitized resistant cells to CP. Mechanistic studies showed that JMJD2 inhibition decreased chromatin association of ATR and Chk1 and inhibited the ATR-Chk1 replication checkpoint. Our results reveal that JMJD2 demethylases are potential therapeutic targets to overcome CP resistance in NSCLC.

Alpha ketoglutarate levels, regulated by p53 and OGDH, determine autophagy and cell fate/apoptosis in response to Nutlin-3a.

Activated p53 can promote apoptosis or cell cycle arrest. Differences in energy metabolism can influence cell fate in response to activated p53. Nutlin-3a is a preclinical drug and small molecule activator of p53. Alpha-ketoglutarate (αKG) levels were reduced in cells sensitive to Nutlin-3a-induced apoptosis and increased in cells resistant to this apoptosis. Add-back of a cell-permeable αKG analog (DMKG) rescued cells from apoptosis in response to Nutlin-3a. OGDH is a component of the αKGDH complex that converts αKG to succinate. OGDH knockdown increased endogenous αKG levels and also rescued cells from Nutlin-3a-induced apoptosis. We previously showed reduced autophagy and ATG gene expression contributes to Nutlin-3a-induced apoptosis. DMKG and OGDH knockdown restored autophagy and ATG gene expression in Nutlin-3a-treated cells. These studies indicate αKG levels, regulated by p53 and OGDH, determine autophagy and apoptosis in response to Nutlin-3a.

Lung epithelial-specific TRIP-1 overexpression maintains epithelial integrity during hyperoxia exposure.

The onset and degree of injury occurring in animals that develop hyperoxic acute lung injury (HALI) is dependent on age at exposure, suggesting that developmentally regulated pathways/factors must underlie initiation of the epithelial injury and subsequent repair. Type II TGFß receptor interacting protein-1 (TRIP-1) is a negative regulator of TGFß signaling, which we have previously shown is a developmentally regulated protein with modulatory effects on epithelial-fibroblastic signaling. The aim of this study was to assess if type II alveolar epithelial cells overexpressing TRIP-1 are protected against hyperoxia-induced epithelial injury, and in turn HALI. Rat lung epithelial cells (RLE) overexpressing TRIP-1 or LacZ were exposed to 85% oxygen for 4 days. A surfactant protein C (SPC)-driven TRIP-1 overexpression mouse (TRIP-1AECTg+ ) was generated and exposed to hyperoxia (>95% for 4 days) at 4 weeks of age to assess the effects TRIP-1 overexpression has on HALI. RLE overexpressing TRIP-1 resisted hyperoxia-induced apoptosis. Mice overexpressing TRIP-1 in their lung type II alveolar epithelial cells (TRIP-1AECTg+ ) showed normal lung development, increased phospho-AKT level and E-cadherin, along with resistance to HALI, as evidence by less TGFß activation, apoptosis, alveolar macrophage influx, KC expression. Taken together, these findings point to existence of a TRIP-1 mediated molecular pathway affording protection against epithelial/acute lung injury.

DZNep represses Bcl-2 expression and modulates apoptosis sensitivity in response to Nutlin-3a.

MDM2 antagonists stabilize and activate wild-type p53, and histone methyltransferase (HMT) inhibitors reduce methylation on histone lysines and arginines. Both MDM2 antagonists and HMT inhibitors are being developed as cancer therapeutics. Wild-type p53 expressing HCT116 colon cancer cells were resistant to apoptosis in response to the MDM2 antagonist Nutlin-3a. However, co-treatment with the HMT inhibitor DZNep sensitized the cells to Nutlin-3a-induced apoptosis. This sensitization resulted from reduced activity of the Bcl-2 gene promoter and a reduction in Bcl-2 mRNA and protein. Surprisingly, DZNep reduced Bcl-2 expression in other colon cancer cell lines (RKO, SW48, and LoVo) but failed to sensitize them to Nutlin-3a. We found these cell lines express elevated levels of Bcl-2 or other Bcl-2-family proteins, including Bcl-xL, Mcl-1, and Bcl-w. Knockdown of Mcl-1 and/or treatment with specific or pan Bcl-2-family inhibitors (BH3 mimetics) sensitized RKO, SW48, and LoVo cells to apoptosis by Nutlin-3a. The results demonstrate 1) DZNep represses the Bcl-2 gene promoter and affects apoptosis sensitivity by reducing Bcl-2 protein expression, and 2) elevated expression of pro-survival Bcl-2 family members protects colon cancer cells from Nutlin-3a-induced apoptosis. Targeting Bcl-2 proteins via DZNep or BH3 mimetics could increase the therapeutic potential of MDM2-antagonists like Nutlin-3a in colon cancer.