Prenatal inflammation remodels lung immunity and function by programming ILC2 hyperactivation.

Here, we examine how prenatal inflammation shapes tissue function and immunity in the lung by reprogramming tissue-resident immune cells from early development. Maternal, but not fetal, type I interferon-mediated inflammation provokes expansion and hyperactivation of group 2 innate lymphoid cells (ILC2s) seeding the developing lung. Hyperactivated ILC2s produce increased IL-5 and IL-13 and are associated with acute Th2 bias, decreased Tregs, and persistent lung eosinophilia into adulthood. ILC2 hyperactivation is recapitulated by adoptive transfer of fetal liver precursors following prenatal inflammation, indicative of developmental programming at the fetal progenitor level. Reprogrammed ILC2 hyperactivation and subsequent lung immune remodeling, including persistent eosinophilia, is concomitant with worsened histopathology and increased airway dysfunction equivalent to papain exposure, indicating increased asthma susceptibility in offspring. Our data elucidate a mechanism by which early-life inflammation results in increased asthma susceptibility in the presence of hyperactivated ILC2s that drive persistent changes to lung immunity during perinatal development.

PAGER-scFGA: unveiling cell functions and molecular mechanisms in cell trajectories through single-cell functional genomics analysis.

Background: Understanding how cells and tissues respond to stress factors and perturbations during disease processes is crucial for developing effective prevention, diagnosis, and treatment strategies. Single-cell RNA sequencing (scRNA-seq) enables high-resolution identification of cells and exploration of cell heterogeneity, shedding light on cell differentiation/maturation and functional differences. Recent advancements in multimodal sequencing technologies have focused on improving access to cell-specific subgroups for functional genomics analysis. To facilitate the functional annotation of cell groups and characterization of molecular mechanisms underlying cell trajectories, we introduce the Pathways, Annotated Gene Lists, and Gene Signatures Electronic Repository for Single-Cell Functional Genomics Analysis (PAGER-scFGA). Results: We have developed PAGER-scFGA, which integrates cell functional annotations and gene-set enrichment analysis into popular single-cell analysis pipelines such as Scanpy. Using differentially expressed genes (DEGs) from pairwise cell clusters, PAGER-scFGA infers cell functions through the enrichment of potential cell-marker genesets. Moreover, PAGER-scFGA provides pathways, annotated gene lists, and gene signatures (PAGs) enriched in specific cell subsets with tissue compositions and continuous transitions along cell trajectories. Additionally, PAGER-scFGA enables the construction of a gene subcellular map based on DEGs and allows examination of the gene functional compartments (GFCs) underlying cell maturation/differentiation. In a real-world case study of mouse natural killer (mNK) cells, PAGER-scFGA revealed two major stages of natural killer (NK) cells and three trajectories from the precursor stage to NK T-like mature stage within blood, spleen, and bone marrow tissues. As the trajectories progress to later stages, the DEGs exhibit greater divergence and variability. However, the DEGs in different trajectories still interact within a network during NK cell maturation. Notably, PAGER-scFGA unveiled cell cytotoxicity, exocytosis, and the response to interleukin (IL) signaling pathways and associated network models during the progression from precursor NK cells to mature NK cells. Conclusion: PAGER-scFGA enables in-depth exploration of functional insights and presents a comprehensive knowledge map of gene networks and GFCs, which can be utilized for future studies and hypothesis generation. It is expected to become an indispensable tool for inferring cell functions and detecting molecular mechanisms within cell trajectories in single-cell studies. The web app (accessible at https://au-singlecell.streamlit.app/) is publicly available.

Gfi1 controls the formation of effector CD8 T cells during chronic infection and cancer.

During chronic infections and tumor progression, CD8 T cells gradually lose their effector functions and become exhausted. These exhausted CD8 T cells are heterogeneous and comprised of different subsets, including self-renewing progenitors that give rise to Ly108 - CX3CR1 + effector-like cells. Generation of these effector-like cells is essential for the control of chronic infections and tumors, albeit limited. However, the precise cues and mechanisms directing the formation and maintenance of exhausted effector-like are incompletely understood. Using genetic mouse models challenged with LCMV Clone 13 or syngeneic tumors, we show that the expression of a transcriptional repressor, growth factor independent 1 (Gfi1) is dynamically regulated in exhausted CD8 T cells, which in turn regulates the formation of exhausted effector-like cells. Gfi1 deletion in T cells dysregulates the chromatin accessibility and transcriptomic programs associated with the differentiation of LCMV Clone 13-specific CD8 T cell exhaustion, preventing the formation of effector-like and terminally exhausted cells while maintaining progenitors and a newly identified Ly108 + CX3CR1 + state. These Ly108 + CX3CR1 + cells have a distinct chromatin profile and may represent an alternative target for therapeutic interventions to combat chronic infections and cancer. In sum, we show that Gfi1 is a critical regulator of the formation of exhausted effector-like cells.

Tumor Microenvironment Landscapes Supporting EGFR-mutant NSCLC Are Modulated at the Single-cell Interaction Level by Unesbulin Treatment.

Lung cancer is the leading cause of cancer deaths. Lethal pulmonary adenocarcinomas (ADC) present with frequent mutations in the EGFR. Genetically engineered murine models of lung cancer expedited comprehension of the molecular mechanisms driving tumorigenesis and drug response. Here, we systematically analyzed the evolution of tumor heterogeneity in the context of dynamic interactions occurring with the intermingled tumor microenvironment (TME) by high-resolution transcriptomics. Our effort identified vulnerable tumor-specific epithelial cells, as well as their cross-talk with niche components (endothelial cells, fibroblasts, and tumor-infiltrating immune cells), whose symbiotic interface shapes tumor aggressiveness and is almost completely abolished by treatment with Unesbulin, a tubulin binding agent that reduces B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) activity. Simultaneous magnetic resonance imaging (MRI) analysis demonstrated decreased tumor growth, setting the stage for future investigations into the potential of novel therapeutic strategies for EGFR-mutant ADCs. SIGNIFICANCE: Targeting the TME is an attractive strategy for treatment of solid tumors. Here we revealed how EGFR-mutant landscapes are affected at the single-cell resolution level during Unesbulin treatment. This novel drug, by targeting cancer cells and their interactions with crucial TME components, could be envisioned for future therapeutic advancements.

A mouse model of Zhu-Tokita-Takenouchi-Kim syndrome reveals indispensable SON functions in organ development and hematopoiesis.

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss of function of SON. While patients with ZTTK syndrome live with numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, including leukopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency shifted cell fate more toward the myeloid lineage but compromised lymphoid lineage development by reducing genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency caused inappropriate activation of erythroid genes and impaired erythropoiesis. These findings highlight the importance of the full gene expression of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

Prenatal inflammation reprograms hyperactive ILC2s that promote allergic lung inflammation and airway dysfunction.

Allergic asthma is a chronic respiratory disease that initiates in early life, but causal mechanisms are poorly understood. Here we examined how prenatal inflammation shapes allergic asthma susceptibility by reprogramming lung immunity from early development. Induction of Type I interferon-mediated inflammation during development provoked expansion and hyperactivation of group 2 innate lymphoid cells (ILC2s) seeding the developing lung. Hyperactivated ILC2s produced increased IL-5 and IL-13, and were associated with acute Th2 bias, eosinophilia, and decreased Tregs in the lung. The hyperactive ILC2 phenotype was recapitulated by adoptive transfer of a fetal liver precursor following exposure to prenatal inflammation, indicative of developmental programming. Programming of ILC2 function and subsequent lung immune remodeling by prenatal inflammation led to airway dysfunction at baseline and in response to papain, indicating increased asthma susceptibility. Our data provide a link by which developmental programming of progenitors by early-life inflammation drives lung immune remodeling and asthma susceptibility through hyperactivation of lung-resident ILC2s. One Sentence Summary: Prenatal inflammation programs asthma susceptibility by inducing the production of hyperactivated ILC2s in the developing lung.

A mouse model of ZTTK syndrome reveals indispensable SON functions in organ development and hematopoiesis.

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hamper our understanding of both SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency inclines cell fate toward the myeloid lineage but compromises lymphoid lineage development by reducing key genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythroid maturation. These findings highlight the importance of the full gene dosage of Son in organ development and hematopoiesis. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

Metabolic adaptation to tyrosine kinase inhibition in leukemia stem cells.

Tyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML-committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the HIF-1-mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.

Hedgehog Signaling Regulates Treg to Th17 Conversion Through Metabolic Rewiring in Breast Cancer.

The tumor immune microenvironment dynamically evolves to support tumor growth and progression. Immunosuppressive regulatory T cells (Treg) promote tumor growth and metastatic seeding in patients with breast cancer. Deregulation of plasticity between Treg and Th17 cells creates an immune regulatory framework that enables tumor progression. Here, we discovered a functional role for Hedgehog (Hh) signaling in promoting Treg differentiation and immunosuppressive activity, and when Hh activity was inhibited, Tregs adopted a Th17-like phenotype complemented by an enhanced inflammatory profile. Mechanistically, Hh signaling promoted O-GlcNAc modifications of critical Treg and Th17 transcription factors, Foxp3 and STAT3, respectively, that orchestrated this transition. Blocking Hh reprogramed Tregs metabolically, dampened their immunosuppressive activity, and supported their transdifferentiation into inflammatory Th17 cells that enhanced the recruitment of cytotoxic CD8+ T cells into tumors. Our results demonstrate a previously unknown role for Hh signaling in the regulation of Treg differentiation and activity and the switch between Tregs and Th17 cells in the tumor microenvironment.

Microenvironmental CXCL12 deletion enhances Flt3-ITD acute myeloid leukemia stem cell response to therapy by reducing p38 MAPK signaling.

Fms-like tyrosine kinase 3 (Flt3) tyrosine kinase inhibitors (Flt3-TKI) have improved outcomes for patients with Flt3-mutated acute myeloid leukemia (AML) but are limited by resistance and relapse, indicating persistence of leukemia stem cells (LSC). Here utilizing a Flt3-internal tandem duplication (Flt3-ITD) and Tet2-deleted AML genetic mouse model we determined that FLT3-ITD AML LSC were enriched within the primitive ST-HSC population. FLT3-ITD LSC showed increased expression of the CXCL12 receptor CXCR4. CXCL12-abundant reticular (CAR) cells were increased in Flt3-ITD AML marrow. CXCL12 deletion from the microenvironment enhanced targeting of AML cells by Flt3-TKI plus chemotherapy treatment, including enhanced LSC targeting. Both treatment and CXCL12 deletion partially reduced p38 mitogen-activated protein kinase (p38) signaling in AML cells and further reduction was seen after treatment in CXCL12 deleted mice. p38 inhibition reduced CXCL12-dependent and -independent maintenance of both murine and human Flt3-ITD AML LSC by MSC and enhanced their sensitivity to treatment. p38 inhibition in combination with chemotherapy plus TKI treatment leads to greater depletion of Flt3-ITD AML LSC compared with CXCL12 deletion. Our studies support roles for CXCL12 and p38 signaling in microenvironmental protection of AML LSC and provide a rationale for inhibiting p38 signaling to enhance Flt3-ITD AML targeting.

Low c-Kit expression identifies primitive, therapy-resistant CML stem cells.

Despite the efficacy of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML), malignant long-term hematopoietic stem cells (LT-HSCs) persist as a source of relapse. However, LT-HSCs are heterogenous and the most primitive, drug-resistant LT-HSC subpopulations are not well characterized. In normal hematopoiesis, self-renewal and long-term reconstitution capacity are enriched within LT-HSCs with low c-Kit expression (c-KITlo). Here, using a transgenic CML mouse model, we found that long-term engraftment and leukemogenic capacity were restricted to c-KITlo CML LT-HSCs. CML LT-HSCs demonstrated enhanced differentiation with expansion of mature progeny following exposure to the c-KIT ligand, stem cell factor (SCF). Conversely, SCF deletion led to depletion of normal LT-HSCs but increase in c-KITlo and total CML LT-HSCs with reduced generation of mature myeloid cells. CML c-KITlo LT-HSCs showed reduced cell cycling and expressed enhanced quiescence and inflammatory gene signatures. SCF administration led to enhanced depletion of CML primitive progenitors but not LT-HSCs after TKI treatment. Human CML LT-HSCs with low or absent c-KIT expression were markedly enriched after TKI treatment. We conclude that CML LT-HSCs expressing low c-KIT levels are enriched for primitive, quiescent, drug-resistant leukemia-initiating cells and represent a critical target for eliminating disease persistence.

WINNER: A network biology tool for biomolecular characterization and prioritization.

Background and contribution: In network biology, molecular functions can be characterized by network-based inference, or "guilt-by-associations." PageRank-like tools have been applied in the study of biomolecular interaction networks to obtain further the relative significance of all molecules in the network. However, there is a great deal of inherent noise in widely accessible data sets for gene-to-gene associations or protein-protein interactions. How to develop robust tests to expand, filter, and rank molecular entities in disease-specific networks remains an ad hoc data analysis process. Results: We describe a new biomolecular characterization and prioritization tool called Weighted In-Network Node Expansion and Ranking (WINNER). It takes the input of any molecular interaction network data and generates an optionally expanded network with all the nodes ranked according to their relevance to one another in the network. To help users assess the robustness of results, WINNER provides two different types of statistics. The first type is a node-expansion p-value, which helps evaluate the statistical significance of adding "non-seed" molecules to the original biomolecular interaction network consisting of "seed" molecules and molecular interactions. The second type is a node-ranking p-value, which helps evaluate the relative statistical significance of the contribution of each node to the overall network architecture. We validated the robustness of WINNER in ranking top molecules by spiking noises in several network permutation experiments. We have found that node degree-preservation randomization of the gene network produced normally distributed ranking scores, which outperform those made with other gene network randomization techniques. Furthermore, we validated that a more significant proportion of the WINNER-ranked genes was associated with disease biology than existing methods such as PageRank. We demonstrated the performance of WINNER with a few case studies, including Alzheimer's disease, breast cancer, myocardial infarctions, and Triple negative breast cancer (TNBC). In all these case studies, the expanded and top-ranked genes identified by WINNER reveal disease biology more significantly than those identified by other gene prioritizing software tools, including Ingenuity Pathway Analysis (IPA) and DiAMOND. Conclusion: WINNER ranking strongly correlates to other ranking methods when the network covers sufficient node and edge information, indicating a high network quality. WINNER users can use this new tool to robustly evaluate a list of candidate genes, proteins, or metabolites produced from high-throughput biology experiments, as long as there is available gene/protein/metabolic network information.

Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.

Autophagy inhibition impairs leukemia stem cell function in FLT3-ITD AML but has antagonistic interactions with tyrosine kinase inhibition.

The FLT3-ITD mutation is associated with poor prognosis in acute myeloid leukemia (AML). FLT3 tyrosine kinase inhibitors (TKIs) demonstrate clinical efficacy but fail to target leukemia stem cells (LSC) and do not generate sustained responses. Autophagy is an important cellular stress response contributing to hematopoietic stem cells (HSC) maintenance and promoting leukemia development. Here we investigated the role of autophagy in regulating FLT3-ITD AML stem cell function and response to TKI treatment. We show that autophagy inhibition reduced quiescence and depleted repopulating potential of FLT3-ITD AML LSC, associated with mitochondrial accumulation and increased oxidative phosphorylation. However, TKI treatment reduced mitochondrial respiration and unexpectedly antagonized the effects of autophagy inhibition on LSC attrition. We further show that TKI-mediated targeting of AML LSC and committed progenitors was p53-dependent, and that autophagy inhibition enhanced p53 activity and increased TKI-mediated targeting of AML progenitors, but decreased p53 activity in LSC and reduced TKI-mediated LSC inhibition. These results provide new insights into the role of autophagy in differentially regulating AML stem and progenitor cells, reveal unexpected antagonistic effects of combined oncogenic tyrosine kinase inhibition and autophagy inhibition in AML LSC, and suggest an alternative approach to target AML LSC quiescence and regenerative potential.

Ex Vivo Expansion of Phenotypic and Transcriptomic Chronic Myeloid Leukemia Stem Cells.

Despite decades of research, standard therapies remain ineffective for most leukemias, pushing toward an essential unmet need for targeted drug screens. Moreover, preclinical drug testing is an important consideration for success of clinical trials without affecting non-transformed stem cells. Using the transgenic chronic myeloid leukemia (CML) mouse model, we determine that leukemic stem cells (LSCs) are transcriptionally heterogenous with a preexistent drug-insensitive signature. To test targeting of potentially important pathways, we establish ex vivo expanded LSCs that have long-term engraftment and give rise to multilineage hematopoiesis. Expanded LSCs share transcriptomic signatures with primary LSCs including enrichment in Wnt, JAK-STAT, MAPK, mTOR and transforming growth factor ß signaling pathways. Drug testing on expanded LSCs show that transforming growth factor ß and Wnt inhibitors had significant effects on the viability of LSCs, but not leukemia-exposed healthy HSCs. This platform allows testing of multiple drugs at the same time to identify vulnerabilities of LSCs.

Deciphering Metabolic Adaptability of Leukemic Stem Cells.

Therapeutic targeting of leukemic stem cells is widely studied to control leukemia. An emerging approach gaining popularity is altering metabolism as a potential therapeutic opportunity. Studies have been carried out on hematopoietic and leukemic stem cells to identify vulnerable pathways without impacting the non-transformed, healthy counterparts. While many metabolic studies have been conducted using stem cells, most have been carried out in vitro or on a larger population of progenitor cells due to challenges imposed by the low frequency of stem cells found in vivo. This creates artifacts in the studies carried out, making it difficult to interpret and correlate the findings to stem cells directly. This review discusses the metabolic difference seen between hematopoietic stem cells and leukemic stem cells across different leukemic models. Moreover, we also shed light on the advancements of metabolic techniques and current limitations and areas for additional research of the field to study stem cell metabolism.

Isocitrate dehydrogenase mutations are associated with altered IL-1ß responses in acute myeloid leukemia.

Mutations in isocitrate dehydrogenase 2 (IDH2) have been noted to impact cellular differentiation in addition to DNA and histone methylation. However, little is known about the impact of IDH2 mutations on intracellular signaling. Using an isogenic cell line model, we investigated both differentiation and signaling responses in IDH2 mutant cells and show augmented responses to inflammatory immune ligands. Using phospho-specific flow and mass cytometry, we demonstrate IDH2 mutant cells were significantly more sensitive to IL-1ß at multiple downstream readouts. Further, bulk RNA sequencing confirmed increases in cytokine-related signaling pathways and NF-κB target genes. Single-cell RNA sequencing of unstimulated and stimulated cells confirmed altered IL-1ß transcriptional responses in the IDH2 mutant cells. Targeted inhibition of the IKK complex reduced IL-1ß responses and induced cell death in primary IDH-mutated leukemia samples. Together, these results confirm altered IL-1ß signaling in IDH2 mutant cells and identify this pathway as a potential therapeutic target.

Serine-threonine Kinase Receptor-Associated Protein is a Critical Mediator of APC Mutation-Induced Intestinal Tumorigenesis Through a Feed-Forward Mechanism.

BACKGROUND & AIMS: Inactivation of the Apc gene is a critical early event in the development of sporadic colorectal cancer (CRC). Expression of serine-threonine kinase receptor-associated protein (STRAP) is elevated in CRCs and is associated with poor outcomes. We investigated the role of STRAP in Apc mutation-induced intestinal tumor initiation and progression. METHODS: We generated Strap intestinal epithelial knockout mice (StrapΔIEC) by crossing mice containing floxed alleles of Strap (Strapfl/fl) with Villin-Cre mice. Then we generated ApcMin/+;Strapfl/fl;Vill-Cre (ApcMin/+;StrapΔIEC) mice for RNA-sequencing analyses to determine the mechanism of function of STRAP. We used human colon cancer cell lines (DLD1, SW480, and HT29) and human and mouse colon tumor-derived organoids for STRAP knockdown and knockout and overexpression experiments. RESULTS: Strap deficiency extended the average survival of ApcMin/+ mice by 80 days and decreased the formation of intestinal adenomas. Expression profiling revealed that the intestinal stem cell signature, the Wnt/ß-catenin signaling, and the MEK/ERK pathway are down-regulated in Strap-deficient adenomas and intestinal organoids. Correlation studies suggest that these STRAP-associated oncogenic signatures are conserved across murine and human colon cancer. STRAP associates with MEK1/2, promotes binding between MEK1/2 and ERK1/2, and subsequently induces the phosphorylation of ERK1/2. STRAP activated Wnt/ß-catenin signaling through MEK/ERK-induced phosphorylation of LRP6. STRAP was identified as a target of mutated Apc and Wnt/ß-catenin signaling as chromatin immunoprecipitation and luciferase assays revealed putative binding sites of the ß-catenin/TCF4 complex on the Strap promoter. CONCLUSIONS: STRAP is a target of, and is required in, Apc mutation/deletion-induced intestinal tumorigenesis through a novel feed-forward STRAP/MEK-ERK/Wnt-ß-catenin/STRAP regulatory axis.