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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.

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications–driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Immune checkpoint inhibitors (ICI) have transformed the treatment of melanoma. However, the majority of patients have primary or acquired resistance to ICIs, limiting durable responses and patient survival. IFNγ signaling and the expression of IFNγ-stimulated genes correlate with either response or resistance to ICIs, in a context-dependent manner. While IFNγ-inducible immunostimulatory genes are required for response to ICIs, chronic IFNγ signaling induces the expression of immunosuppressive genes, promoting resistance to these therapies. Here, we show that high levels of Unc-51 like kinase 1 (ULK1) correlate with poor survival in patients with melanoma and overexpression of ULK1 in melanoma cells enhances IFNγ-induced expression of immunosuppressive genes, with minimal effects on the expression of immunostimulatory genes. In contrast, genetic or pharmacologic inhibition of ULK1 reduces expression of IFNγ-induced immunosuppressive genes. ULK1 binds IRF1 in the nuclear compartment of melanoma cells, controlling its binding to the programmed death-ligand 1 promoter region. In addition, pharmacologic inhibition of ULK1 in combination with anti-programmed cell death protein 1 therapy further reduces melanoma tumor growth in vivo. Our data suggest that targeting ULK1 represses IFNγ-dependent immunosuppression. These findings support the combination of ULK1 drug-targeted inhibition with ICIs for the treatment of patients with melanoma to improve response rates and patient outcomes. Implications: This study identifies ULK1, activated downstream of IFNγ signaling, as a druggable target to overcome resistance mechanisms to ICI therapy in metastatic melanoma.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinasessup1-5/sup. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUTamp;Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

With advances in molecular medicine and precision approaches, there has been significant improvement in the treatment of acute myeloid leukemia (AML) in recent years. This reflects better understanding of molecular and metabolic pathways in leukemia cells, including BCL2 upregulation that prevents apoptosis, FLT3 tyrosine kinase activating mutations that allow uncontrolled proliferation, and IDH mutations that result in differentiation block.We performed a compressive review of important pre-clinical studies in AML that involve major molecular and metabolic pathways in AML, and we discussed standard therapeutic modalities and ongoing clinical trials for patients with AML, as well as an overall update of recent efforts in this area.Targeting these pathways has resulted in improvement in the overall survival of some groups of AML patients. Secondary AML and TP53 mutated AML remain challenging subtypes of AML with limited treatment options and represent areas of unmet research need. Ongoing work with menin inhibitors in MLL rearranged leukemia, which comprise a large portion of secondary AML cases, the development of CAR T cell products and targeting the CD47 receptor on macrophages in myeloid neoplasms including in TP53 mutated AML have provided hope for these challenging subtypes of AML.

Inhibition of CHAF1B increases IFNα-induced anticlonogenic effects in MPN. HEL cells (A) and SET-2 cells (B) were transfected with either control (Ctrl) siRNA or CHAF1B siRNA, and leukemic CFU-L colony formation was assessed in clonogenic assays in the presence or absence of IFNα, as indicated. Data are expressed as percentage of colony formation over control siRNA-transfected untreated cells (control), and the scatter dot plots represent means ± SEM of four independent experiments for each cell line. C, Clonogenic capability of primary malignant erythroid progenitors isolated from PV patients transfected with either control (Ctrl) siRNA or CHAF1B siRNA and either left untreated or treated with IFNα, as indicated. Data are expressed as percentage of colony formation over control siRNA-transfected untreated cells (control) and shown are means ± SEM of six independent experiments using cells from six different patients with PV. Percentages for the same patient are represented by the same symbol and different patients have different symbols. A–C, Statistical analyses were performed using one-way ANOVA followed by Tukey multiple comparisons test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

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.

Gene-targeted inhibition of CHAF1B increases IFNα-inducible mRNA expression of ISGs in JAK2V617F-positive leukemic cells. qRT-PCR analyses of the indicated genes in control (Ctrl) siRNA or CHAF1B siRNA-transfected JAK2V617F-positive HEL (A) and SET-2 (B) cells, either left untreated or treated with IFNα for 6 hours. GAPDH mRNA expression was used for normalization. Data are expressed as fold change over control siRNA-transfected untreated cells (control) and represent means ± SEM of three independent experiments for each cell line. Statistical analyses were performed using one-way ANOVA followed by Tukey multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, ChIP assay was performed in untreated and IFNα-treated (for 6 hours at 5,000 IU/mL) HEL cells at the ISG15 promoter and the IFIT1 promoter for CHAF1B binding using an anti-CHAF1B antibody. IgG antibody was used for each promoter region as negative control. Scatter dot plots show data as percent enrichment relative to input ± SEM for three independent experiments. Statistical analyses were performed using one-way ANOVA followed by Tukey multiple comparisons test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. D, Immunoblot analysis of ULK1 expression in control siRNA (Ctrlsi) and ULK1 siRNA (ULK1si) transfected HEL cells either left untreated or treated with IFNα (for 6 hours at 5,000 IU/mL), as indicated. GAPDH levels were used as loading control. Blots are representative of three independent experiments used to perform ChIP assays shown in E. E, ChIP assay was performed in untreated (UT) and IFNα-treated control (Ctrl) siRNA- and ULK1 siRNA-transfected HEL cells at the ISG15 promoter and the IFIT1 promoter for CHAF1B binding using an anti-CHAF1B antibody. F, HEL cells were pretreated for 1 hour with either DMSO (Ctrl and IFNα groups) or SBI-0206965 (SBI; 10 μmol/L; SBI and SBI+IFNα groups) followed by 6 hours of treatment with either DMSO (Ctrl), SBI (10 μmol/L), IFNα (5,000 IU/mL) or SBI+IFNα, as indicated. ChIP assay was performed in HEL cells at the ISG15 promoter and the IFIT1 promoter for CHAF1B binding using an anti-CHAF1B antibody. E and F, IgG antibody was used for each promoter region as negative control. Scatter dot plots show data as percent enrichment relative to input ± SEM for three independent experiments. Statistical analyses were performed using two-way ANOVA followed by Tukey multiple comparisons test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Relevant statistical differences are shown for the binding of CHAF1B to ISG15 and IFIT1 promoter regions between experimental conditions.

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

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.

Metastasis is the cause of over 90% of all deaths associated with breast cancer, yet the strategies to predict cancer spreading based on primary tumor profiles and therefore prevent metastasis are egregiously limited. As rare precursor cells to metastasis, circulating tumor cells (CTCs) in multicellular clusters in the blood are 20-50 times more likely to produce viable metastasis than single CTCs. However, the molecular mechanisms underlying various CTC clusters, such as homotypic tumor cell clusters and heterotypic tumor-immune cell clusters, are yet to be fully elucidated. Combining machine learning-assisted computational ranking with experimental demonstration to assess cell adhesion candidates, we identified a transmembrane protein Plexin- B2 (PB2) as a new therapeutic target that drives the formation of both homotypic and heterotypic CTC clusters. High PB2 expression in human primary tumors predicts an unfavorable distant metastasis-free survival and is enriched in CTC clusters compared to single CTCs in advanced breast cancers. Loss of PB2 reduces formation of homotypic tumor cell clusters as well as heterotypic tumor-myeloid cell clusters in triple-negative breast cancer. Interactions between PB2 and its ligand Sema4C on tumor cells promote homotypic cluster formation, and PB2 binding with Sema4A on myeloid cells (monocytes) drives heterotypic CTC cluster formation, suggesting that metastasizing tumor cells hijack the PB2/Sema family axis to promote lung metastasis in breast cancer. Additionally, using a global proteomic analysis, we identified novel downstream effectors of the PB2 pathway associated with cancer stemness, cell cycling, and tumor cell clustering in breast cancer. Thus, PB2 is a novel therapeutic target for preventing new metastasis.

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

Supplementary figures

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 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 NFκB target genes, consistent with a negative regulatory role for SLFN11 on the NFκB pathway. Furthermore, our studies identify p21 as a direct transcriptional target of NFκB2 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.Significance:We identify a negative regulatory role for SLFN11 in noncanonical NFκB signaling that results in suppression of the cell-cycle inhibitor p21. We provide evidence that SLFN11 contributes to regulation of stem cell markers in GBM, promoting the malignant phenotype. In addition, SLFN11 targeting triggers p21 expression and antitumor responses. Our studies define a highly novel function for SLFN11 and identify it as a potential therapeutic target for GBM.

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.

The potent immunosuppression induced by glioblastoma (GBM) is one of the primary obstacles to finding effective immunotherapies. One hallmark of the GBM-associated immunosuppressive landscape is the massive infiltration of myeloid-derived suppressor cells (MDSC) and, to a lesser extent, regulatory T cells (Treg) within the tumor microenvironment. Here, we showed that regulatory B cells (Breg) are a prominent feature of the GBM microenvironment in both preclinical models and clinical samples. Forty percent of GBM patients (n = 60) scored positive for B-cell tumor infiltration. Human and mouse GBM-associated Bregs were characterized by immunosuppressive activity toward activated CD8+ T cells, the overexpression of inhibitory molecules PD-L1 and CD155, and production of immunosuppressive cytokines TGFβ and IL10. Local delivery of B cell–depleting anti-CD20 immunotherapy improved overall survival of animals (IgG vs. anti-CD20 mean survival: 18.5 vs. 33 days, P = 0.0001), suggesting a potential role of Bregs in GBM progression. We unveiled that GBM-associated MDSCs promoted regulatory B-cell function by delivering microvesicles transporting membrane-bound PD-L1, able to be up-taken by tumoral B cells. The transfer of functional PD-L1 via microvesicles conferred Bregs the potential to suppress CD8+ T-cell activation and acquisition of an effector phenotype. This work uncovered the role of B cells in GBM physiopathology and provides a mechanism by which the GBM microenvironment controls B cell–mediated immunosuppression.See related Spotlight on p. 1902

Supplementary Tables and Supplementary Figure Legends

S1. Overexpression of ULK1 in melanoma cells has minor effects on transcription of IFNγ-induced immunostimulatory genes.S2. Effects of gene targeted inhibition of ULK1 in transcription of IFNγ-induced immunostimulatory genes in melanoma cells.S3. Effects of drug-targeted inhibition of ULK1 in transcription of IFNγ-induced immunostimulatory genes in melanoma cells.S4. Effects of siRNA-mediated knockdown of ULK1 on IFNγ-inducedSTAT1 phosphorylation.S5. Identification of putative cytoplasmic and nuclear ULK1 binding partners in melanoma cells upon IFNγ stimulation by mass spectrometry analysis.S6. Pharmacological inhibition of ULK1 enhances the anti-tumor effects of anti-PD-1 therapy in YUMMER1.7 mouse melanoma in vivo model.S7. Tumor infiltrating CD8+ T cells and TAMCs in vehicle-isotype-treated mice.S8. Tumor infiltrating CD8+ T cells and TAMCs in ULK inhibitor-treated mice.S9. Tumor infiltrating CD8+ T cells and TAMCs in anti-PD-1-treated mice.S10. Tumor infiltrating CD8+ T cells and TAMCs in ULKi plus anti-PD-1-treated mice.S11. Tumor infiltrating CD4+ T cells and Tregs in vehicle-isotype-treated mice.S12. Tumor infiltrating CD4+ T cells and Tregs in ULK inhibitor-treated mice.S13. Tumor infiltrating CD4+ T cells and Tregs in anti-PD-1treated mice.S14. Tumor infiltrating CD4+ T cells and Tregs in ULKi plus anti-PD-1-treated mice.S15. Tumor infiltrating CD8+ T cells and TAMCs in control, ULKi and/or anti-PD-1-treated mice.S16. Tumor infiltrating CD4+ T cells and Tregs in control, ULKi and/or anti-PD-1-treated mice.S17. Gating strategy for flow cytometric immunophenotyping of mouse melanoma tumors.

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.

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 p110β 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.

Pairwise tests for differences in stem cell frequencies.

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.

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.

Immune checkpoint inhibitors (ICI) have transformed the treatment of melanoma. However, the majority of patients have primary or acquired resistance to ICIs, limiting durable responses and patient survival. IFNγ signaling and the expression of IFNγ-stimulated genes correlate with either response or resistance to ICIs, in a context-dependent manner. While IFNγ-inducible immunostimulatory genes are required for response to ICIs, chronic IFNγ signaling induces the expression of immunosuppressive genes, promoting resistance to these therapies. Here, we show that high levels of Unc-51 like kinase 1 (ULK1) correlate with poor survival in patients with melanoma and overexpression of ULK1 in melanoma cells enhances IFNγ-induced expression of immunosuppressive genes, with minimal effects on the expression of immunostimulatory genes. In contrast, genetic or pharmacologic inhibition of ULK1 reduces expression of IFNγ-induced immunosuppressive genes. ULK1 binds IRF1 in the nuclear compartment of melanoma cells, controlling its binding to the programmed death-ligand 1 promoter region. In addition, pharmacologic inhibition of ULK1 in combination with anti-programmed cell death protein 1 therapy further reduces melanoma tumor growth in vivo. Our data suggest that targeting ULK1 represses IFNγ-dependent immunosuppression. These findings support the combination of ULK1 drug-targeted inhibition with ICIs for the treatment of patients with melanoma to improve response rates and patient outcomes.Implications:This study identifies ULK1, activated downstream of IFNγ signaling, as a druggable target to overcome resistance mechanisms to ICI therapy in metastatic melanoma.

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

Human pancreatic ductal adenocarcinoma (PDAC) tumors are associated with dysregulation of mRNA translation. In this report, it is demonstrated that PDAC cells grown in collagen exhibit increased activation of the MAPK-interacting protein kinases (MNK) that mediate eIF4E phosphorylation. Pharmacologic and genetic targeting of MNKs reverse epithelial–mesenchymal transition (EMT), decrease cell migration, and reduce protein expression of the EMT-regulator ZEB1 without affecting ZEB1 mRNA levels. Paradoxically, targeting eIF4E, the best-characterized effector of MNKs, increases ZEB1 mRNA expression through repression of ZEB1-targeting miRNAs, miR-200c and miR-141. In contrast, targeting the MNK effector hnRNPA1, which can function as a translational repressor, increases ZEB1 protein without increasing ZEB1 mRNA levels. Importantly, treatment with MNK inhibitors blocks growth of chemoresistant PDAC cells in collagen and decreases the number of aldehyde dehydrogenase activity–positive (Aldefluor+) cells. Significantly, MNK inhibitors increase E-cadherin mRNA levels and decrease vimentin mRNA levels in human PDAC organoids without affecting ZEB1 mRNA levels. Importantly, MNK inhibitors also decrease growth of human PDAC organoids.Implications: These results demonstrate differential regulation of ZEB1 and EMT by MNKs and eIF4E, and identify MNKs as potential targets in pancreatic cancer. Mol Cancer Res; 14(2); 216–27. ©2015 AACR.

Supplementary Figure Legends. Supplementary Figure 1: Targeted disruption of AMPKα1/2 does not affect arsenicinducible phosphorylation of AKT at Ser473. Supplementary Figure 2: As- Biotin binds to AMPKα as measured by mass spectroscopy

List of the putative ULK1 interactor proteins identified by mass spectrometry analysis in A375 cells

Mesenchymal (MES) and proneural (PN) are two distinct glioma stem cell (GSC) populations that drive therapeutic resistance in glioblastoma (GBM). We screened a panel of 650 small molecules against patient-derived GBM cells to discover compounds targeting specific GBM subtypes. Arsenic trioxide (ATO), an FDA-approved drug that crosses the blood–brain barrier, was identified as a potent PN-specific compound in the initial screen and follow-up validation studies. Furthermore, MES and PN GSCs exhibited differential sensitivity to ATO. As ATO has been shown to activate the MAPK-interacting kinase 1 (MNK1)-eukaryotic translation initiation factor 4E (eIF4E) pathway and subsequent mRNA translation in a negative regulatory feedback manner, the mechanistic role of ATO resistance in MES GBM was explored. In GBM cells, ATO-activated translation initiation cellular events via the MNK1–eIF4E signaling axis. Furthermore, resistance to ATO in intracranial PDX tumors correlated with high eIF4E phosphorylation. Polysomal fractionation and microarray analysis of GBM cells were performed to identify ATO's effect on mRNA translation and enrichment of anti-apoptotic mRNAs in the ATO-induced translatome was found. Additionally, it was determined that MNK inhibition sensitized MES GSCs to ATO in neurosphere and apoptosis assays. Finally, examination of the effect of ATO on patients from a phase I/II clinical trial of ATO revealed that PN GBM patients responded better to ATO than other subtypes as demonstrated by longer overall and progression-free survival.Implications: These findings raise the possibility of a unique therapeutic approach for GBM, involving MNK1 targeting to sensitize MES GSCs to drugs like arsenic trioxide. Mol Cancer Res; 16(1); 32–46. ©2017 AACR.

Supplemental Table 1. Genes and rearrangements studied; Supplemental Table 2: Genes added over time to Guardant360 assay; Supplemental Table 3. Defining concordance and partial concordance for a particular gene; Supplemental Table 4. Concordance and partial concordance among only genes with genomic alterations in either assay; Supplemental Table 5. Sensitivity, specificity, and diagnostic accuracy across 5 genes, including variants of unknown significance (VUS)

Body weight of mice from flank tumor xenograft experiment

Relates to materials & methods.

Supplemental Figure 2. Glioblastoma patient overall survival.

Knockdown of Angpt1 does not lead to inhibition of leukemic cell viability

MDS Patient Characteristics

Supplemental Figure 5. Confirmation of gut microbiota depletion.