Merestinib blocks Mnk kinase activity in acute myeloid leukemia progenitors and exhibits antileukemic effects in vitro and in vivo.

Mitogen-activated protein kinase interacting protein kinases (Mnks) play important roles in the development and progression of acute myeloid leukemia (AML) by regulating eukaryotic translation initiation factor 4E (eIF4E) activation. Inhibiting Mnk1/2-induced phosphorylation of eIF4E may represent a unique approach for the treatment of AML. We provide evidence for antileukemic effects of merestinib, an orally bioavailable multikinase inhibitor with suppressive effects on Mnk activity. Our studies show that merestinib effectively blocks eIF4E phosphorylation in AML cells and suppresses primitive leukemic progenitors from AML patients in vitro and in an AML xenograft model in vivo. Our findings provide evidence for potent preclinical antileukemic properties of merestinib and support its clinical development for the treatment of patients with AML.

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.

Discovery of a distinct domain in cyclin A sufficient for centrosomal localization independently of Cdk binding.

Centrosomes have recently emerged as key regulators of the cell cycle. The G1/S transition requires a functional centrosome, and centrosomal localization of numerous proteins, including cyclin/Cdk complexes, is important for the G2/M transition. Here we identify a modular centrosomal localization signal (CLS) localizing cyclin A to centrosomes independently of Cdk binding. The cyclin A CLS is located in a distinct part of the molecule compared with the cyclin E CLS and includes the MRAIL hydrophobic patch involved in substrate recognition. The cyclin A CLS interacts with p27(KIP1), and expression of p27(KIP1) removes cyclin A but not cyclin E from centrosomes. Expression of the cyclin A CLS displaces both endogenous cyclin A and E from centrosomes and inhibits DNA replication, supporting an emerging concept that DNA replication is linked to centrosomal events. Structural analysis indicates that differences in surface charge and length of the C-terminal helix explain why the MRAIL region in cyclin E is not a functional CLS. These results indicate that the cyclin A CLS may contribute to targeting and recognition of centrosomal Cdk substrates and is required for specific effects of p27(KIP1) on cyclin A-Cdk2.

Kicking off the polo game.

Polo-like kinase 1 (Plk1) is essential for checkpoint recovery and the activation of key mitotic enzymes; however, its own activation mechanism has remained elusive. Recent findings show that Bora, a G(2)-M expressed protein, facilitates Plk1 activation by the oncogenic kinase Aurora A in G(2). During mitosis, Plk1-dependent Bora degradation promotes Aurora A localization to the centrosome and/or spindle. Bora-dependent regulation provides important new insights into interactions between key mitotic kinases.

Emerging Role of Glioma Stem Cells in Mechanisms of Therapy Resistance.

Since their discovery at the beginning of this millennium, glioma stem cells (GSCs) have sparked extensive research and an energetic scientific debate about their contribution to glioblastoma (GBM) initiation, progression, relapse, and resistance. Different molecular subtypes of GBM coexist within the same tumor, and they display differential sensitivity to chemotherapy. GSCs contribute to tumor heterogeneity and recapitulate pathway alterations described for the three GBM subtypes found in patients. GSCs show a high degree of plasticity, allowing for interconversion between different molecular GBM subtypes, with distinct proliferative potential, and different degrees of self-renewal and differentiation. This high degree of plasticity permits adaptation to the environmental changes introduced by chemo- and radiation therapy. Evidence from mouse models indicates that GSCs repopulate brain tumors after therapeutic intervention, and due to GSC plasticity, they reconstitute heterogeneity in recurrent tumors. GSCs are also inherently resilient to standard-of-care therapy, and mechanisms of resistance include enhanced DNA damage repair, MGMT promoter demethylation, autophagy, impaired induction of apoptosis, metabolic adaptation, chemoresistance, and immune evasion. The remarkable oncogenic properties of GSCs have inspired considerable interest in better understanding GSC biology and functions, as they might represent attractive targets to advance the currently limited therapeutic options for GBM patients. This has raised expectations for the development of novel targeted therapeutic approaches, including targeting GSC plasticity, chimeric antigen receptor T (CAR T) cells, and oncolytic viruses. In this review, we focus on the role of GSCs as drivers of GBM and therapy resistance, and we discuss how insights into GSC biology and plasticity might advance GSC-directed curative approaches.

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.

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.

Modeling Therapy-Driven Evolution of Glioblastoma with Patient-Derived Xenografts.

Adult-type diffusely infiltrating gliomas, of which glioblastoma is the most common and aggressive, almost always recur after treatment and are fatal. Improved understanding of therapy-driven tumor evolution and acquired therapy resistance in gliomas is essential for improving patient outcomes, yet the majority of the models currently used in preclinical research are of therapy-naïve tumors. Here, we describe the development of therapy-resistant IDH-wildtype glioblastoma patient-derived xenografts (PDX) through orthotopic engraftment of therapy naïve PDX in athymic nude mice, and repeated in vivo exposure to the therapeutic modalities most often used in treating glioblastoma patients: radiotherapy and temozolomide chemotherapy. Post-temozolomide PDX became enriched for C>T transition mutations, acquired inactivating mutations in DNA mismatch repair genes (especially MSH6), and developed hypermutation. Such post-temozolomide PDX were resistant to additional temozolomide (median survival decrease from 80 days in parental PDX to 42 days in a temozolomide-resistant derivative). However, temozolomide-resistant PDX were sensitive to lomustine (also known as CCNU), a nitrosourea which induces tumor cell apoptosis by a different mechanism than temozolomide. These PDX models mimic changes observed in recurrent GBM in patients, including critical features of therapy-driven tumor evolution. These models can therefore serve as valuable tools for improving our understanding and treatment of recurrent glioma.

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.

Discovery of a signaling feedback circuit that defines interferon responses in myeloproliferative neoplasms.

Interferons (IFNs) are key initiators and effectors of the immune response against malignant cells and also directly inhibit tumor growth. IFNα is highly effective in the treatment of myeloproliferative neoplasms (MPNs), but the mechanisms of action are unclear and it remains unknown why some patients respond to IFNα and others do not. Here, we identify and characterize a pathway involving PKCδ-dependent phosphorylation of ULK1 on serine residues 341 and 495, required for subsequent activation of p38 MAPK. We show that this pathway is essential for IFN-suppressive effects on primary malignant erythroid precursors from MPN patients, and that increased levels of ULK1 and p38 MAPK correlate with clinical response to IFNα therapy in these patients. We also demonstrate that IFNα treatment induces cleavage/activation of the ULK1-interacting ROCK1/2 proteins in vitro and in vivo, triggering a negative feedback loop that suppresses IFN responses. Overexpression of ROCK1/2 is seen in MPN patients and their genetic or pharmacological inhibition enhances IFN-anti-neoplastic responses in malignant erythroid precursors from MPN patients. These findings suggest the clinical potential of pharmacological inhibition of ROCK1/2 in combination with IFN-therapy for the treatment of MPNs.

Spindle pole regulation by a discrete Eg5-interacting domain in TPX2.

Targeting protein for Xklp2 (TPX2) activates the Ser/Thr kinase Aurora A in mitosis and targets it to the mitotic spindle [1, 2]. These effects on Aurora A are mediated by the N-terminal domain of TPX2, whereas a C-terminal fragment has been reported to affect microtubule nucleation [3]. Using the Xenopus system, we identified a novel role of TPX2 during mitosis. Injection of TPX2 or its C terminus (TPX2-CT) into blastomeres of two-cell embryos led to potent cleavage arrest. Despite cleavage arrest, TPX2-injected embryos biochemically undergo multiple rounds of DNA synthesis and mitosis, and arrested blastomeres have abnormal spindles, clustered centrosomes, and an apparent failure of cytokinesis. In Xenopus S3 cells, transfection of TPX2-FL causes spindle collapse, whereas TPX2-CT blocks pole segregation, resulting in apposing spindle poles with no evident displacement of Aurora A. Analysis of TPX2-CT deletion peptides revealed that only constructs able to interact with the class 5 kinesin-like motor protein Eg5 induce the spindle phenotypes. Importantly, injection of Eg5 into TPX2-CT-arrested blastomeres causes resumption of cleavage. These results define a discrete domain within the C terminus of TPX2 that exerts a novel Eg5-dependent function in spindle pole segregation.

Therapeutic targeting of transcriptional elongation in diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is associated with transcriptional dysregulation driven by H3K27 mutation. The super elongation complex (SEC) is required for transcriptional elongation through release of RNA polymerase II (Pol II). Inhibition of transcription elongation by SEC disruption can be an effective therapeutic strategy of H3K27M-mutant DIPG. Here, we tested the effect of pharmacological disruption of the SEC in H3K27M-mutant DIPG to advance understanding of the molecular mechanism and as a new therapeutic strategy for DIPG. METHODS: Short hairpin RNAs (shRNAs) were used to suppress the expression of AF4/FMR2 4 (AFF4), a central SEC component, in H3K27M-mutant DIPG cells. A peptidomimetic lead compound KL-1 was used to disrupt a functional component of SEC. Cell viability assay, colony formation assay, and apoptosis assay were utilized to analyze the effects of KL-1 treatment. RNA- and ChIP-sequencing were used to determine the effects of KL-1 on gene expression and chromatin occupancy. We treated mice bearing H3K27M-mutant DIPG patient-derived xenografts (PDXs) with KL-1. Intracranial tumor growth was monitored by bioluminescence image and therapeutic response was evaluated by animal survival. RESULTS: Depletion of AFF4 significantly reduced the cell growth of H3K27M-mutant DIPG. KL-1 increased genome-wide Pol II occupancy and suppressed transcription involving multiple cellular processes that promote cell proliferation and differentiation of DIPG. KL-1 treatment suppressed DIPG cell growth, increased apoptosis, and prolonged animal survival with H3K27M-mutant DIPG PDXs. CONCLUSIONS: SEC disruption by KL-1 increased therapeutic benefit in vitro and in vivo, supporting a potential therapeutic activity of KL-1 in H3K27M-mutant DIPG.

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.

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.

Pharmacological mTOR targeting enhances the antineoplastic effects of selective PI3Kα inhibition in medulloblastoma.

Despite recent advances in the treatment of medulloblastoma, patients in high-risk categories still face very poor outcomes. Evidence indicates that a subpopulation of cancer stem cells contributes to therapy resistance and tumour relapse in these patients. To prevent resistance and relapse, the development of treatment strategies tailored to target subgroup specific signalling circuits in high-risk medulloblastomas might be similarly important as targeting the cancer stem cell population. We have previously demonstrated potent antineoplastic effects for the PI3Kα selective inhibitor alpelisib in medulloblastoma. Here, we performed studies aimed to enhance the anti-medulloblastoma effects of alpelisib by simultaneous catalytic targeting of the mTOR kinase. Pharmacological mTOR inhibition potently enhanced the suppressive effects of alpelisib on cancer cell proliferation, colony formation and apoptosis and additionally blocked sphere-forming ability of medulloblastoma stem-like cancer cells in vitro. We identified the HH effector GLI1 as a target for dual PI3Kα and mTOR inhibition in SHH-type medulloblastoma and confirmed these results in HH-driven Ewing sarcoma cells. Importantly, pharmacologic mTOR inhibition greatly enhanced the inhibitory effects of alpelisib on medulloblastoma tumour growth in vivo. In summary, these findings highlight a key role for PI3K/mTOR signalling in GLI1 regulation in HH-driven cancers and suggest that combined PI3Kα/mTOR inhibition may be particularly interesting for the development of effective treatment strategies in high-risk medulloblastomas.

Potent Antineoplastic Effects of Combined PI3Kα-MNK Inhibition in Medulloblastoma.

Medulloblastoma is a highly malignant pediatric brain tumor associated with poor outcome. Developing treatments that target the cancer stem cell (CSC) population in medulloblastoma are important to prevent tumor relapse and induce long-lasting clinical responses. We utilized medulloblastoma neurospheres that display CSC characteristics and found activation of the PI3K/AKT pathway in sphere-forming cells. Of all class IA PI3Ks, only the PI3Kα isoform was required for sphere formation by medulloblastoma cells. Knockdown of p110α, but not p110ß or p110δ, significantly disrupted cancer stem cell frequencies as determined by extreme limiting dilution analysis (ELDA), indicating an essential role for the PI3Kα catalytic isoform in medulloblastoma CSCs. Importantly, pharmacologic inhibition of the MAPK-interacting kinase (MNK) enhanced the antineoplastic effects of targeted PI3Kα inhibition in medulloblastoma. This indicates that MNK signaling promotes survival in medulloblastoma, suggesting dual PI3Kα and MNK inhibition may provide a novel approach to target and eliminate medulloblastoma CSCs. We also observed a significant reduction in tumor formation in subcutaneous and intracranial mouse xenograft models, which further suggests that this combinatorial approach may represent an efficient therapeutic strategy for medulloblastoma. IMPLICATIONS: These findings raise the possibility of a unique therapeutic approach for medulloblastoma, involving MNK targeting to sensitize medulloblastoma CSCs to PI3Kα inhibition.

Inhibitory effects of SEL201 in acute myeloid leukemia.

MAPK interacting kinase (MNK), a downstream effector of mitogen-activated protein kinase (MAPK) pathways, activates eukaryotic translation initiation factor 4E (eIF4E) and plays a key role in the mRNA translation of mitogenic and antiapoptotic genes in acute myeloid leukemia (AML) cells. We examined the antileukemic properties of a novel MNK inhibitor, SEL201. Our studies provide evidence that SEL201 suppresses eIF4E phosphorylation on Ser209 in AML cell lines and in primary patient-derived AML cells. Such effects lead to growth inhibitory effects and leukemic cell apoptosis, as well as suppression of leukemic progenitor colony formation. Combination of SEL201 with 5'-azacytidine or rapamycin results in synergistic inhibition of AML cell growth. Collectively, these results suggest that SEL201 has significant antileukemic activity and further underscore the relevance of the MNK pathway in leukemogenesis.

Polo-like kinase 1: target and regulator of anaphase-promoting complex/cyclosome-dependent proteolysis.

Polo-like kinase 1 (Plk1) is a key regulator of progression through mitosis. Although Plk1 seems to be dispensable for entry into mitosis, its role in spindle formation and exit from mitosis is crucial. Recent evidence suggests that a major role of Plk1 in exit from mitosis is the regulation of inhibitors of the anaphase-promoting complex/cyclosome (APC/C), such as the early mitotic inhibitor 1 (Emi1) and spindle checkpoint proteins. Thus, Plk1 and the APC/C control mitotic regulators by both phosphorylation and targeted ubiquitylation to ensure the fidelity of chromosome separation at the metaphase to anaphase transition. The mechanisms underlying the control of genomic stability by Plk1 are discussed in this review.

Antineoplastic effects of selective CDK9 inhibition with atuveciclib on cancer stem-like cells in triple-negative breast cancer.

Treatment options for triple-negative breast cancer (TNBC) are limited due to the lack of efficient targeted therapies, frequently resulting in recurrence and metastatic disease. Accumulating evidence suggests that a small population of cancer stem-like cells (CSLCs) is responsible for tumor recurrence and therapy resistance. Here we investigated the role of cyclin-dependent kinase 9 (CDK9) in TNBC. Using The Cancer Genome Atlas (TCGA) data we found high-CDK9 expression correlates with worse overall survival in TNBC patients. Pharmacologic inhibition of CDK9 with atuveciclib in high-CDK9 expressing TNBC cell lines reduced expression of CDK9 targets MYC and MCL1 and decreased cell proliferation and survival. Importantly, atuveciclib inhibited the growth of mammospheres and reduced the percentage of CD24low/CD44high cells, indicating disruption of breast CSLCs (BCSLCs). Furthermore, atuveciclib impaired 3D invasion of tumorspheres suggesting inhibition of both invasion and metastatic potential. Finally, atuveciclib enhanced the antineoplastic effects of Cisplatin and promoted inhibitory effects on BCSLCs grown as mammospheres. Together, these findings suggest CDK9 as a potential therapeutic target in aggressive forms of CDK9-high TNBC.

HDL nanoparticles targeting sonic hedgehog subtype medulloblastoma.

Medulloblastoma is the most common paediatric malignant brain cancer and there is a need for new targeted therapeutic approaches to more effectively treat these malignant tumours, which can be divided into four molecular subtypes. Here, we focus on targeting sonic hedgehog (SHH) subtype medulloblastoma, which accounts for approximately 25% of all cases. The SHH subtype relies upon cholesterol signalling for tumour growth and maintenance of tumour-initiating cancer stem cells (CSCs). To target cholesterol signalling, we employed biomimetic high-density lipoprotein nanoparticles (HDL NPs) which bind to the HDL receptor, scavenger receptor type B-1 (SCARB1), depriving cells of natural HDL and their cholesterol cargo. We demonstrate uptake of HDL NPs in SCARB1 expressing medulloblastoma cells and depletion of cholesterol levels in cancer cells. HDL NPs potently blocked proliferation of medulloblastoma cells, as well as hedgehog-driven Ewing sarcoma cells. Furthermore, HDL NPs disrupted colony formation in medulloblastoma and depleted CSC populations in medulloblastoma and Ewing sarcoma. Altogether, our findings provide proof of principle for the development of a novel targeted approach for the treatment of medulloblastoma using HDL NPs. These findings present HDL-mimetic nanoparticles as a promising therapy for sonic hedgehog (SHH) subtype medulloblastoma and possibly other hedgehog-driven cancers.