MNK activity is regulated by the p38 and Erk MAPK pathways. Phosphorylation of MNK leads to its activation and binding to the eIF4G/eIF4E complex. MNK then phosphorylates eIF4E at Ser209, whose activation is associated with oncogene translation, leading to tumorigenesis. Given this important role for eIF4E in tumorigenesis, MNK inhibition with novel small molecule inhibitors could be a promising strategy to combat AML, which continues to be an area of unmet medical need. Here, we report the medicinal optimization of a series of novel inhibitors and their evaluation of their effects on eIF4E and leukemia cell viability. We discovered a class of ether-containing compounds with a high MNK1/2 selectivity. These MNK inhibitors show good potency in reducing cell viability and colony formation and have desirable pharmacokinetic properties. X-ray cocrystallization was accomplished to confirm the binding mode of our inhibitors and aid in future optimization.

Abstract Glioblastoma multiforme (GBM) or grade 4 IDHWT glioma is the most malignant brain tumor with a median survival of ~15 months. Current treatment options are limited to a combination of surgery, radiation, and the alkylating agent temozolomide (TMZ). However, despite this aggressive treatment regimen, patients inevitably develop recurrent tumors, which depict a high degree of resistance to DNA damage induced by TMZ and irradiation. Therefore, it is imperative to better understand cellular signals contributing to GBM tumorigenesis and therapy resistance. We have previously established potential roles for members of the protein family of schlafens (SLFNs) in GBM. SLFNs have established roles in cell differentiation, cell proliferation, immune responses, and an expanding role in cancer biology. Several human SLFNs have been linked to chemosensitivity in various cancer types. In particular, SLFN5 mRNA levels progressively increased from grade 2 to grade 4 glioma suggesting a possible therapeutic target for GBM. Therefore, in an effort to better understand the role of SLFN5 in GBM tumorigenesis, CRISPR/Cas9-mediated SLFN5 knockout glioblastoma cell lines and PDX lines were generated. Cells lacking expression of SLFN5 showed increased DNA damage signaling, as judged by elevated basal levels of the DNA damage marker γH2AX and persistent phosphorylation of Chk2. Gene ontology analysis indicated increased expression of p53 pathway components in SLFN5 knockout cells. In GBM, p53 activity is commonly deregulated due to high mutations in the p53 pathway. In line with this, SLFN5 knockout cells depicted increased sensitivity to the mutant p53 reactivator APR-246 correlating with p53 mutational status. APR-246 induced PARP cleavage in SLFN5 knockout cells was rescued by the ROS scavenger N-acetyl-D-cysteine (NAC). Our results suggest increased DNA damage signaling in SLFN5 deficient cells. Furthermore, mutant p53 reactivator APR-246 induced apoptosis is dependent on ROS signaling. In summary, we provide evidence for a protective role of SLFN5 to limit DNA damage and ROS signaling in GBM. Citation Format: Ricardo E. Perez, Frank Eckerdt, Leonidas C. Platanias. Disruption of SLFN5 enhances vulnerability to APR-246, a mutant p53 reactivator in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A008.

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.

This table shows the initial cell viability screen in GBM cells. Relates to Figure 1C.

Biological processes in which putative ULK1-protein complexes are involved in the cytosol.

Supplementary Table S2 lists putative SLFN11 binding partners detected after untreated and/or irradiated cells.

These figures show the PN and MES specific drugs identified by the follow-up cell viability screen. Also shown are the enrichment of translation gene sets in MES GSCs. Relates to Figure 1.

Fig. S1: Expression of SLFN family members after SLFN11 knockout. Fig. S2: Reduced 3-D invasion after SLFN11 knockout. Fig. S3: Expression of stem/progenitor markers after SLFN11 add-back. Table S1: Key resources table.

CHAF1B interacts with ULK1 in the nuclear compartment of JAK2V617F-positive cells and is overexpressed in patients with MPN. A and B, Left, ULK1–protein complexes were co-IP using an anti-ULK1 antibody from cytosolic and nuclear protein fractions isolated from untreated or IFNα-treated (10 or 240 minutes) HEL (A) or SET-2 (B) cells and then resolved by SDS-PAGE. As control, cytosolic and nuclear lysates isolated from cells treated with IFNα for 240 minutes were incubated with normal rabbit IgG (RIgG) antibody. Interaction between ULK1 and CHAF1B was assessed by immunoblotting with anti-ULK1 and anti-CHAF1B antibodies. Note: ǂ, unspecific band. A and B, Right, Equal amounts of cytosolic and nuclear protein lysates isolated from untreated and IFNα-treated HEL (A) and SET-2 (B) cells used for co-IPs were resolved by SDS-PAGE, transferred to PVDF membranes and then immunoblotted with antibodies against ULK1, CHAF1B, α-tubulin (cytosolic marker), and lamin A/C (nuclear marker), as indicated. A and B, Blots are representative of three independent experiments. C, Scatter dot plot of log2CHAF1B mRNA expression in neutrophils from healthy individuals (normal, n = 11) and patients with ET (n = 47), PMF (n = 18), and PV (n = 28). Data were extracted from NCBI GEO: GSE54646 study (21) and analyzed using GraphPad Prim 8. Shown are means ± SEM. Statistical analysis was performed using one-way ANOVA followed by Dunnett multiple comparisons test to assess P values between patients with MPN and healthy individuals. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Scatter dot plots of log2CHAF1B mRNA expression in neutrophils from patients with ET (green triangles), PMF (purple diamonds), and PV (red squares) carrying wild-type (WT) or mutant (MUT) JAK2 (D), CALR (E), and TET2 (F) genes (JAK2 WT: ET n = 20, PMF n = 10, PV n = 5; JAK2 MUT: ET n = 26, PMF n = 8, PV n = 23; CALR WT: ET n = 27, PMF n = 11, PV n = 26; CALR MUT: ET n = 15, PMF n = 5, PV n = 1; TET2 WT: ET n = 45, PMF n = 17, PV n = 24; TET2 MUT: ET n = 2, PMF n = 1, PV n = 4). Data were extracted from NCBI GEO: GSE54646 study (21) and analyzed using GraphPad Prim 8. Patients for which no information was available for the mutational status for the JAK2, CALR, or TET2 genes were excluded from the analysis. Shown are means ± SEM. Statistical analyses were performed using two-sample two-tailed t test: *, P < 0.05.

Pairwise tests for differences in stem cell frequencies.

Supplementary materials and methods for ALDEFLUOR assay and CD44 staining and data showing that patient-derived GSCs are enriched for ALDH+ cells and express CD44.

Supplementary Table S4 lists proteins that were found to bind SLFN11 after irradiation.

This table shows genes enriched in untreated and ATO-treated polysomes. Relates to Figure 4D.

Body weight of mice from flank tumor xenograft experiment

Supplemental data related to Figs. 2 and 5 S1. Collagen-dependent MNK phosphorylation in chemoresistant PDAC cells. S2. Targeting eIF4E increases ZEB1 levels in AsPC1 and Panc1 cells. S3. eIF4E and MNK1/2 do not regulate expression of primary transcript of miR-200 (pri-miR-200) microRNAs A and B. S4. miR-141 and miR-200c regulate ZEB1 levels in CD18-CR cells A. S5. Targeting the MNK effector hnRNPA1 increases ZEB1 protein without increasing ZEB1 mRNA levels.

Relates to materials & methods.

Supplementary Table S3 lists proteins that were found to bind SLFN11 before or after irradiation.

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.

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.