Stress-triggered hematopoietic stem cell proliferation relies on PrimPol-mediated repriming.

Stem cell division is linked to tumorigenesis by yet-elusive mechanisms. The hematopoietic system reacts to stress by triggering hematopoietic stem and progenitor cell (HSPC) proliferation, which can be accompanied by chromosomal breakage in activated hematopoietic stem cells (HSCs). However, whether these lesions persist in their downstream progeny and induce a canonical DNA damage response (DDR) remains unclear. Inducing HSPC proliferation by simulated viral infection, we report that the associated DNA damage is restricted to HSCs and that proliferating HSCs rewire their DDR upon endogenous and clastogen-induced damage. Combining transcriptomics, single-cell and single-molecule assays on murine bone marrow cells, we found accelerated fork progression in stimulated HSPCs, reflecting engagement of PrimPol-dependent repriming, at the expense of replication fork reversal. Ultimately, competitive bone marrow transplantation revealed the requirement of PrimPol for efficient HSC amplification and bone marrow reconstitution. Hence, fine-tuning replication fork plasticity is essential to support stem cell functionality upon proliferation stimuli.

Engaging community members in cancer research: an assessment of an NCI-designated cancer center.

PURPOSE: Despite the importance of engaging community members in research, multiple barriers exist. We conducted a mixed-methods evaluation to understand the opportunities and challenges of engaging community members in basic, clinical, translational, and population science research. METHODS: We designed a survey and an interview guide based on the constructs of the Consolidated Framework for Implementation Research. Surveys were distributed electronically to all cancer center investigators and interviews were conducted virtually with a select group of basic, clinical, and population science investigators. Survey data (n = 77) were analyzed across all respondents using frequency counts and mean scores; bivariate analyses examined differences in responses by research program affiliation, gender, race, and faculty rank. Interviews (n = 16) were audio recorded, transcribed verbatim, and analyzed using a reflective thematic approach. RESULTS: There was strong agreement among investigators that "Community engagement in research will help the SKCC address cancer disparities in the catchment area" (M 4.2, SD 0.9) and less agreement with items such as "I know how to find and connect with community members who I can engage in my research" (M 2.5, SD 1.3). Investigators mentioned challenges in communicating complex science to a lay audience but were open to training and workshops to acquire skills needed to integrate community members into their research. CONCLUSION: Cancer centers should develop and promote training and collaborative opportunities for investigators and community members. Overcoming challenges will lead to more patient- and community-centered cancer research in the future.

Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC.

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at ∼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors.

Interaction of the oncoprotein transcription factor MYC with its chromatin cofactor WDR5 is essential for tumor maintenance.

The oncoprotein transcription factor MYC is overexpressed in the majority of cancers. Key to its oncogenic activity is the ability of MYC to regulate gene expression patterns that drive and maintain the malignant state. MYC is also considered a validated anticancer target, but efforts to pharmacologically inhibit MYC have failed. The dependence of MYC on cofactors creates opportunities for therapeutic intervention, but for any cofactor this requires structural understanding of how the cofactor interacts with MYC, knowledge of the role it plays in MYC function, and demonstration that disrupting the cofactor interaction will cause existing cancers to regress. One cofactor for which structural information is available is WDR5, which interacts with MYC to facilitate its recruitment to chromatin. To explore whether disruption of the MYC-WDR5 interaction could potentially become a viable anticancer strategy, we developed a Burkitt's lymphoma system that allows replacement of wild-type MYC for mutants that are defective for WDR5 binding or all known nuclear MYC functions. Using this system, we show that WDR5 recruits MYC to chromatin to control the expression of genes linked to biomass accumulation. We further show that disrupting the MYC-WDR5 interaction within the context of an existing cancer promotes rapid and comprehensive tumor regression in vivo. These observations connect WDR5 to a core tumorigenic function of MYC and establish that, if a therapeutic window can be established, MYC-WDR5 inhibitors could be developed as anticancer agents.

IL-33 Is a Cell-Intrinsic Regulator of Fitness during Early B Cell Development.

IL-33 is an IL-1 family member protein that is a potent driver of inflammatory responses in both allergic and nonallergic disease. This proinflammatory effect is mediated primarily by extracellular release of IL-33 from stromal cells and binding of the C-terminal domain of IL-33 to its receptor ST2 on targets such as CD4+ Th2 cells, ILC2, and mast cells. Notably, IL-33 has a distinct N-terminal domain that mediates nuclear localization and chromatin binding. However, a defined in vivo cell-intrinsic role for IL-33 has not been established. We identified IL-33 expression in the nucleus of progenitor B (pro-B) and large precursor B cells in the bone marrow, an expression pattern unique to B cells among developing lymphocytes. The IL-33 receptor ST2 was not expressed within the developing B cell lineage at either the transcript or protein level. RNA sequencing analysis of wild-type and IL-33-deficient pro-B and large precursor B cells revealed a unique, IL-33-dependent transcriptional profile wherein IL-33 deficiency led to an increase in E2F targets, cell cycle genes, and DNA replication and a decrease in the p53 pathway. Using mixed bone marrow chimeric mice, we demonstrated that IL-33 deficiency resulted in an increased frequency of developing B cells via a cell-intrinsic mechanism starting at the pro-B cell stage paralleling IL-33 expression. Finally, IL-33 was detectable during early B cell development in humans and IL33 mRNA expression was decreased in B cell chronic lymphocytic leukemia samples compared with healthy controls. Collectively, these data establish a cell-intrinsic, ST2-independent role for IL-33 in early B cell development.

The proto-oncogene function of Mdm2 in bone.

Mouse double minute 2 (Mdm2) is a multifaceted oncoprotein that is highly regulated with distinct domains capable of cellular transformation. Loss of Mdm2 is embryonically lethal, making it difficult to study in a mouse model without additional genetic alterations. Global overexpression through increased Mdm2 gene copy number (Mdm2Tg ) results in the development of hematopoietic neoplasms and sarcomas in adult animals. In these mice, we found an increase in osteoblastogenesis, differentiation, and a high bone mass phenotype. Since it was difficult to discern the cell lineage that generated this phenotype, we generated osteoblast-specific Mdm2 overexpressing (Mdm2TgOb ) mice in 2 different strains, C57BL/6 and DBA. These mice did not develop malignancies; however, these animals and the MG63 human osteosarcoma cell line with high levels of Mdm2 showed an increase in bone mineralization. Importantly, overexpression of Mdm2 corrected age-related bone loss in mice, providing a role for the proto-oncogenic activity of Mdm2 in bone health of adult animals.

SMARCAL1 maintains telomere integrity during DNA replication.

The SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1) DNA translocase is one of several related enzymes, including ZRANB3 (zinc finger, RAN-binding domain containing 3) and HLTF (helicase-like transcription factor), that are recruited to stalled replication forks to promote repair and restart replication. These enzymes can perform similar biochemical reactions such as fork reversal; however, genetic studies indicate they must have unique cellular activities. Here, we present data showing that SMARCAL1 has an important function at telomeres, which present an endogenous source of replication stress. SMARCAL1-deficient cells accumulate telomere-associated DNA damage and have greatly elevated levels of extrachromosomal telomere DNA (C-circles). Although these telomere phenotypes are often found in tumor cells using the alternative lengthening of telomeres (ALT) pathway for telomere elongation, SMARCAL1 deficiency does not yield other ALT phenotypes such as elevated telomere recombination. The activity of SMARCAL1 at telomeres can be separated from its genome-maintenance activity in bulk chromosomal replication because it does not require interaction with replication protein A. Finally, this telomere-maintenance function is not shared by ZRANB3 or HLTF. Our results provide the first identification, to our knowledge, of an endogenous source of replication stress that requires SMARCAL1 for resolution and define differences between members of this class of replication fork-repair enzymes.

Concordant dysregulation of miR-5p and miR-3p arms of the same precursor microRNA may be a mechanism in inducing cell proliferation and tumorigenesis: a lung cancer study.

A precursor microRNA (miRNA) has two arms: miR-5p and miR-3p (miR-5p/-3p). Depending on the tissue or cell types, both arms can become functional. However, little is known about their coregulatory mechanisms during the tumorigenic process. Here, by using the large-scale miRNA expression profiles of five cancer types, we revealed that several of miR-5p/-3p arms were concordantly dysregulated in each cancer. To explore possible coregulatory mechanisms of concordantly dysregulated miR-5p/-3p pairs, we developed a robust computational framework and applied it to lung cancer data. The framework deciphers miR-5p/-3p coregulated protein interaction networks critical to lung cancer development. As a novel part in the method, we uniquely applied the second-order partial correlation to minimize false-positive regulations. Using 279 matched miRNA and mRNA expression profiles extracted from tumor and normal lung tissue samples, we identified 17 aberrantly expressed miR-5p/-3p pairs that potentially modulate the gene expression of 35 protein complexes. Functional analyses revealed that these complexes are associated with cancer-related biological processes, suggesting the oncogenic potential of the reported miR-5p/-3p pairs. Specifically, we revealed that the reduced expression of miR-145-5p/-3p pair potentially contributes to elevated expression of genes in the "FOXM1 transcription factor network" pathway, which may consequently lead to uncontrolled cell proliferation. Subsequently, the regulation of miR-145-5p/-3p in the FOXM1signaling pathway was validated by a cohort of 104 matched miRNA and protein (reverse-phase protein array) expression profiles in lung cancer. In summary, our computational framework provides a novel tool to study miR-5p/-3p coregulatory mechanisms in cancer and other diseases.

Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides.

The pathogenesis of mycosis fungoides (MF), the most common cutaneous T-cell lymphoma (CTCL), is unknown. Although genetic alterations have been identified, none are considered consistently causative in MF. To identify potential drivers of MF, we performed whole-genome sequencing of MF tumors and matched normal skin. Targeted ultra-deep sequencing of MF samples and exome sequencing of CTCL cell lines were also performed. Multiple mutations were identified that affected the same pathways, including epigenetic, cell-fate regulation, and cytokine signaling, in MF tumors and CTCL cell lines. Specifically, interleukin-2 signaling pathway mutations, including activating Janus kinase 3 (JAK3) mutations, were detected. Treatment with a JAK3 inhibitor significantly reduced CTCL cell survival. Additionally, the mutation data identified 2 other potential contributing factors to MF, ultraviolet light, and a polymorphism in the tumor suppressor p53 (TP53). Therefore, genetic alterations in specific pathways in MF were identified that may be viable, effective new targets for treatment.

Reproducible combinatorial regulatory networks elucidate novel oncogenic microRNAs in non-small cell lung cancer.

While previous studies reported aberrant expression of microRNAs (miRNAs) in non-small cell lung cancer (NSCLC), little is known about which miRNAs play central roles in NSCLC's pathogenesis and its regulatory mechanisms. To address this issue, we presented a robust computational framework that integrated matched miRNA and mRNA expression profiles in NSCLC using feed-forward loops. The network consists of miRNAs, transcription factors (TFs), and their common predicted target genes. To discern the biological meaning of their associations, we introduced the direction of regulation. A network edge validation strategy using three independent NSCLC expression profiling data sets pinpointed reproducible biological regulations. Reproducible regulation, which may reflect the true molecular interaction, has not been applied to miRNA-TF co-regulatory network analyses in cancer or other diseases yet. We revealed eight hub miRNAs that connected to a higher proportion of targets validated by independent data sets. Network analyses showed that these miRNAs might have strong oncogenic characteristics. Furthermore, we identified a novel miRNA-TF co-regulatory module that potentially suppresses the tumor suppressor activity of the TGF-ß pathway by targeting a core pathway molecule (TGFBR2). Follow-up experiments showed two miRNAs (miR-9-5p and miR-130b-3p) in this module had increased expression while their target gene TGFBR2 had decreased expression in a cohort of human NSCLC. Moreover, we demonstrated these two miRNAs directly bind to the 3' untranslated region of TGFBR2. This study enhanced our understanding of miRNA-TF co-regulatory mechanisms in NSCLC. The combined bioinformatics and validation approach we described can be applied to study other types of diseases.

The Mdm network and its regulation of p53 activities: a rheostat of cancer risk.

The potent transcriptional activity of p53 (Trp53, TP53) must be kept in check for normal cell growth and survival. Tumors, which drastically deviate from these parameters, have evolved multiple mechanisms to inactivate TP53, the most prevalent of which is the emergence of TP53 missense mutations, some of which have gain-of-function activities. Another important mechanism by which tumors bypass TP53 functions is via increased levels of two TP53 inhibitors, MDM2, and MDM4. Studies in humans and in mice reveal the complexity of TP53 regulation and the exquisite sensitivity of this pathway to small changes in regulation. Here, we summarize the factors that impinge on TP53 activity and thus cell death/arrest or tumor development.

Glycolytic rate and lymphomagenesis depend on PARP14, an ADP ribosyltransferase of the B aggressive lymphoma (BAL) family.

Poly(ADP-ribose)polymerase (PARP)14--a member of the B aggressive lymphoma (BAL) family of macrodomain-containing PARPs--is an ADP ribosyltransferase that interacts with Stat6, enhances induction of certain genes by IL-4, and is expressed in B lymphocytes. We now show that IL-4 enhancement of glycolysis in B cells requires PARP14 and that this process is central to a role of PARP14 in IL-4-induced survival. Thus, enhancements of AMP-activated protein kinase activity restored both IL-4-induced glycolytic activity in Parp14(-/-) B cells and prosurvival signaling by this cytokine. Suppression of apoptosis is central to B-lymphoid oncogenesis, and elevated macro-PARP expression has been correlated with lymphoma aggressiveness. Strikingly, PARP14 deficiency delayed B lymphomagenesis and reversed the block to B-cell maturation driven by the Myc oncogene. Collectively, these findings reveal links between a mammalian ADP ribosyltransferase, cytokine-regulated metabolic activity, and apoptosis; show that PARP14 influences Myc-induced oncogenesis; and suggest that the PARP14-dependent capacity to increase cellular metabolic rates may be an important determinant of lymphoma pathobiology.

Murine double minute 2: p53-independent roads lead to genome instability or death.

The oncoprotein murine double minute 2 (Mdm2) is frequently overexpressed in many types of human malignancies. Although Mdm2 has an essential role in negatively regulating the p53 tumor suppressor, it also has less well characterized p53-independent functions that influence pathways that are crucial for controlling tumorigenesis. In addition to the impact Mdm2 has on p53-independent apoptosis, mounting evidence is linking increased Mdm2 levels to altered cell-cycle regulation, DNA replication and DNA repair leading to loss of genome stability. Mdm2 involvement in pathways that influence chromosome stability and cell death, distinct from its role in the p53 pathway, strengthens the position of Mdm2 as a desirable therapeutic target for the treatment of human cancers.

Identifying Targetable Vulnerabilities to Circumvent or Overcome Venetoclax Resistance in Diffuse Large B-Cell Lymphoma.

Clinical trials with single-agent venetoclax/ABT-199 (anti-apoptotic BCL2 inhibitor) revealed that diffuse large B-cell lymphoma (DLBCL) is not solely dependent on BCL2 for survival. Gaining insight into pathways/proteins that increase venetoclax sensitivity or unique vulnerabilities in venetoclax-resistant DLBCL would provide new potential treatment avenues. Therefore, we generated acquired venetoclax-resistant DLBCL cells and evaluated these together with intrinsically venetoclax-resistant and -sensitive DLBCL lines. We identified resistance mechanisms, including alterations in BCL2 family members that differed between intrinsic and acquired venetoclax resistance and increased dependencies on specific pathways. Although combination treatments with BCL2 family member inhibitors may overcome venetoclax resistance, RNA-sequencing and drug/compound screens revealed that venetoclax-resistant DLBCL cells, including those with TP53 mutation, had a preferential dependency on oxidative phosphorylation. Mitochondrial electron transport chain complex I inhibition induced venetoclax-resistant, but not venetoclax-sensitive, DLBCL cell death. Inhibition of IDH2 (mitochondrial redox regulator) synergistically overcame venetoclax resistance. Additionally, both acquired and intrinsic venetoclax-resistant DLBCL cells were similarly sensitive to inhibitors of transcription, B-cell receptor signaling, and class I histone deacetylases. These approaches were also effective in DLBCL, follicular, and marginal zone lymphoma patient samples. Our results reveal there are multiple ways to circumvent or overcome the diverse venetoclax resistance mechanisms in DLBCL and other B-cell lymphomas and identify critical targetable pathways for future clinical investigations.

Decoding the lncRNAome Across Diverse Cellular Stresses Reveals Core p53-effector Pan-cancer Suppressive lncRNAs.

Despite long non-coding RNAs (lncRNAs) emerging as key contributors to malignancies, their transcriptional regulation, tissue-type expression under different conditions, and functions remain largely unknown. Developing a combined computational and experimental framework, which integrates pan-cancer RNAi/CRISPR screens, and genomic, epigenetic, and expression profiles (including single-cell RNA sequencing), we report across multiple cancers, core p53-transcriptionally regulated lncRNAs, which were thought to be primarily cell/tissue-specific. These lncRNAs were consistently directly transactivated by p53 with different cellular stresses in multiple cell types and associated with pan-cancer cell survival/growth suppression and patient survival. Our prediction results were verified through independent validation datasets, our own patient cohort, and cancer cell experiments. Moreover, a top predicted p53-effector tumor-suppressive lncRNA (we termed PTSL) inhibited cell proliferation and colony formation by modulating the G2 regulatory network, causing G2 cell-cycle arrest. Therefore, our results elucidated previously unreported, high-confidence core p53-targeted lncRNAs that suppress tumorigenesis across cell types and stresses. Significance: Identification of pan-cancer suppressive lncRNAs transcriptionally regulated by p53 across different cellular stresses by integrating multilayered high-throughput molecular profiles. This study provides critical new insights into the p53 tumor suppressor by revealing the lncRNAs in the p53 cell-cycle regulatory network and their impact on cancer cell growth and patient survival.

Targeted MDM2 Degradation Reveals a New Vulnerability for p53-Inactivated Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) frequently inactivate p53, increasing their aggressiveness and therapy resistance. We identified an unexpected protein vulnerability in p53-inactivated TNBC and designed a new PROteolysis TArgeting Chimera (PROTAC) to target it. Our PROTAC selectively targets MDM2 for proteasome-mediated degradation with high-affinity binding and VHL recruitment. MDM2 loss in p53 mutant/deleted TNBC cells in two-dimensional/three-dimensional culture and TNBC patient explants, including relapsed tumors, causes apoptosis while sparing normal cells. Our MDM2-PROTAC is stable in vivo, and treatment of TNBC xenograft-bearing mice demonstrates tumor on-target efficacy with no toxicity to normal cells, significantly extending survival. Transcriptomic analyses revealed upregulation of p53 family target genes. Investigations showed activation and a required role for TAp73 to mediate MDM2-PROTAC-induced apoptosis. Our data, challenging the current MDM2/p53 paradigm, show MDM2 is required for p53-inactivated TNBC cell survival, and PROTAC-targeted MDM2 degradation is an innovative potential therapeutic strategy for TNBC and superior to existing MDM2 inhibitors. SIGNIFICANCE: p53-inactivated TNBC is an aggressive, therapy-resistant, and lethal breast cancer subtype. We designed a new compound targeting an unexpected vulnerability we identified in TNBC. Our MDM2-targeted degrader kills p53-inactivated TNBC cells, highlighting the requirement for MDM2 in TNBC cell survival and as a new therapeutic target for this disease. See related commentary by Peuget and Selivanova, p. 1043. This article is highlighted in the In This Issue feature, p. 1027.

4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.

Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.

MTBP and MYC: A Dynamic Duo in Proliferation, Cancer, and Aging.

The oncogenic transcription factor c-MYC (MYC) is highly conserved across species and is frequently overexpressed or dysregulated in human cancers. MYC regulates a wide range of critical cellular and oncogenic activities including proliferation, metabolism, metastasis, apoptosis, and differentiation by transcriptionally activating or repressing the expression of a large number of genes. This activity of MYC is not carried out in isolation, instead relying on its association with a myriad of protein cofactors. We determined that MDM Two Binding Protein (MTBP) indirectly binds MYC and is a novel MYC transcriptional cofactor. MTBP promotes MYC-mediated transcriptional activity, proliferation, and cellular transformation by binding in a protein complex with MYC at MYC-bound promoters. This discovery provided critical context for data linking MTBP to aging as well as a rapidly expanding body of evidence demonstrating MTBP is overexpressed in many human malignancies, is often linked to poor patient outcomes, and is necessary for cancer cell survival. As such, MTBP represents a novel and potentially broad reaching oncologic drug target, particularly when MYC is dysregulated. Here we have reviewed the discovery of MTBP and the initial controversy with its function as well as its associations with proliferation, MYC, DNA replication, aging, and human cancer.

Systematic lncRNA mapping to genome-wide co-essential modules uncovers cancer dependency on uncharacterized lncRNAs.

Quantification of gene dependency across hundreds of cell lines using genome-scale CRISPR screens has revealed co-essential pathways/modules and critical functions of uncharacterized genes. In contrast to protein-coding genes, robust CRISPR-based loss-of-function screens are lacking for long noncoding RNAs (lncRNAs), which are key regulators of many cellular processes, leaving many essential lncRNAs unidentified and uninvestigated. Integrating copy number, epigenetic, and transcriptomic data of >800 cancer cell lines with CRISPR-derived co-essential pathways, our method recapitulates known essential lncRNAs and predicts proliferation/growth dependency of 289 poorly characterized lncRNAs. Analyzing lncRNA dependencies across 10 cancer types and their expression alteration by diverse growth inhibitors across cell types, we prioritize 30 high-confidence pan-cancer proliferation/growth-regulating lncRNAs. Further evaluating two previously uncharacterized top proliferation-suppressive lncRNAs (PSLR-1, PSLR-2) showed they are transcriptionally regulated by p53, induced by multiple cancer treatments, and significantly correlate to increased cancer patient survival. These lncRNAs modulate G2 cell cycle-regulating genes within the FOXM1 transcriptional network, inducing a G2 arrest and inhibiting proliferation and colony formation. Collectively, our results serve as a powerful resource for exploring lncRNA-mediated regulation of cellular fitness in cancer, circumventing current limitations in lncRNA research.

CTpathway: a CrossTalk-based pathway enrichment analysis method for cancer research.

BACKGROUND: Pathway enrichment analysis (PEA) is a common method for exploring functions of hundreds of genes and identifying disease-risk pathways. Moreover, different pathways exert their functions through crosstalk. However, existing PEA methods do not sufficiently integrate essential pathway features, including pathway crosstalk, molecular interactions, and network topologies, resulting in many risk pathways that remain uninvestigated. METHODS: To overcome these limitations, we develop a new crosstalk-based PEA method, CTpathway, based on a global pathway crosstalk map (GPCM) with >440,000 edges by combing pathways from eight resources, transcription factor-gene regulations, and large-scale protein-protein interactions. Integrating gene differential expression and crosstalk effects in GPCM, we assign a risk score to genes in the GPCM and identify risk pathways enriched with the risk genes. RESULTS: Analysis of >8300 expression profiles covering ten cancer tissues and blood samples indicates that CTpathway outperforms the current state-of-the-art methods in identifying risk pathways with higher accuracy, reproducibility, and speed. CTpathway recapitulates known risk pathways and exclusively identifies several previously unreported critical pathways for individual cancer types. CTpathway also outperforms other methods in identifying risk pathways across all cancer stages, including early-stage cancer with a small number of differentially expressed genes. Moreover, the robust design of CTpathway enables researchers to analyze both bulk and single-cell RNA-seq profiles to predict both cancer tissue and cell type-specific risk pathways with higher accuracy. CONCLUSIONS: Collectively, CTpathway is a fast, accurate, and stable pathway enrichment analysis method for cancer research that can be used to identify cancer risk pathways. The CTpathway interactive web server can be accessed here http://www.jianglab.cn/CTpathway/ . The stand-alone program can be accessed here https://github.com/Bioccjw/CTpathway .