optimized-mouse-model-to-induce-colitis-associated-colon-cancer-using

Optimized Mouse Model to Induce Colitis-Associated Colon Cancer using Azoxymethane and Dextran Sulfate Sodium for Tumor Immunology and Therapy Studies

Azoxymethane (AOM, a DNA-damaging agent) and dextran sulfate sodium (DSS, a colitis-promoting agent) can induce tumors in the large intestine of mice with ...

University of Florida College of Medicine

University of Florida Health Cancer Center

FQI1: a transcription-methylation switch for cancer.

Somatic mutations in DNMT3A are recurrent events across a range of blood cancers. Dnmt3a loss of function in hematopoietic stem cells (HSCs) skews divisions toward self-renewal at the expense of differentiation. Moreover, DNMT3A mutations can be detected in the blood of aging individuals, indicating that mutant cells outcompete normal HSCs over time. It is important to understand how these mutations provide a competitive advantage to HSCs. Here we show that Dnmt3a-null HSCs can regenerate over at least 12 transplant generations in mice, far exceeding the lifespan of normal HSCs. Molecular characterization reveals that this in vivo immortalization is associated with gradual and focal losses of DNA methylation at key regulatory regions associated with self-renewal genes, producing a highly stereotypical HSC phenotype in which epigenetic features are further buttressed. These findings lend insight into the preponderance of DNMT3A mutations in clonal hematopoiesis and the persistence of mutant clones after chemotherapy.

Loss of Dnmt3a Immortalizes Hematopoietic Stem Cells In Vivo.

DNMT3A alterations associated with myeloid malignancies dictate differential responses to hypomethylating agents.

In the last decade, large-scale genomic studies in patients with hematologic malignancies identified recurrent somatic alterations in epigenetic modifier genes. Among these, the  DNA methyltransferase  has emerged as one of the most frequently mutated genes in adult myeloid as well as lymphoid malignancies and in clonal hematopoiesis. In this review, we discuss recent advances in our understanding of the biochemical and structural consequences of  mutations on DNA methylation catalysis and binding interactions and summarize their effects on epigenetic patterns and gene expression changes implicated in the pathogenesis of hematologic malignancies. We then review the role played by mutant  in clonal hematopoiesis, accompanied by its effect on immune cell function and inflammatory responses. Finally, we discuss how this knowledge informs therapeutic approaches for hematologic malignancies with mutant .

Alterations to  in Hematologic Malignancies.

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c). NPM1c maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c-associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c-driven leukemogenesis by rectifying the signature of NPM1c leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1) mice. HoxBlincTg and Npm1 HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c AML.

HOXBLINC long non-coding RNA activation promotes leukemogenesis in NPM1-mutant acute myeloid leukemia.

In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations.

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.

Cytarabine and other chain-terminating nucleoside analogs that damage replication forks in rapidly proliferating cells are a cornerstone of leukemia chemotherapy, yet the outcomes remain unsatisfactory because of resistance and toxicity. Better understanding of DNA damage repair and downstream effector mechanisms in different disease subtypes can guide combination strategies that sensitize leukemia cells to cytarabine without increasing side effects. We have previously found that mutations in DNMT3A, one of the most commonly mutated genes in acute myeloid leukemia and associated with poor prognosis, predisposed cells to DNA damage and cell killing by cytarabine, cladribine, and other nucleoside analogs, which coincided with PARP1 dysfunction and DNA repair defect (Venugopal K, Feng Y, Nowialis P, et al. Clin Cancer Res 2022;28:756-769). In this article, we first overview DNA repair mechanisms that remove aberrant chain-terminating nucleotides as determinants of sensitivity or resistance to cytarabine and other nucleoside analogs. Next, we discuss PARP inhibition as a rational strategy to increase cytarabine efficacy in cells without DNMT3A mutations, while considering the implications of PARP inhibitor resistance for promoting clonal hematopoiesis. Finally, we examine the utility of p53 potentiators to boost leukemia cell killing by cytarabine in the context of mutant DNMT3A. Systematic profiling of DNA damage repair proficiency has the potential to uncover subtype-specific therapeutic dependencies in AML.

Combination strategies to promote sensitivity to cytarabine-induced replication stress in acute myeloid leukemia with and without DNMT3A mutations.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of mutated preleukemic cells. Individuals with CH are at an increased risk of developing hematopoietic malignancies. Here, we describe a novel animal model carrying a recurrent TET2 missense mutation frequently found in patients with CH and leukemia. In a fashion similar to CH, animals show signs of disease late in life when they develop a wide range of myeloid neoplasms, including acute myeloid leukemia (AML). Using single-cell transcriptomic profiling of the bone marrow, we show that disease progression in aged animals correlates with an enhanced inflammatory response and the emergence of an aberrant inflammatory monocytic cell population. The gene signature characteristic of this inflammatory population is associated with poor prognosis in patients with AML. Our study illustrates an example of collaboration between a genetic lesion found in CH and inflammation, leading to transformation and the establishment of blood neoplasms.

The Impact of Inflammation-Induced Tumor Plasticity during Myeloid Transformation.

DNMT3A mutations confer a poor prognosis in acute myeloid leukemia (AML), but the molecular mechanisms downstream of DNMT3A mutations in disease pathogenesis are not completely understood, limiting targeted therapeutic options. The role of miRNA in DNMT3A-mutant AML pathogenesis is understudied.

miR-196b-TLR7/8 Signaling Axis Regulates Innate Immune Signaling and Myeloid Maturation in DNMT3A-Mutant AML.

C-terminal mutation of Nucleophosmin 1 (NPM1) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, molecular mechanisms underlying NPM1-driven leukemogenesis remain elusive. Here, we report that NPM1 activates signature HOX genes and reprograms cell cycle regulators by altering CTCF-driven topologically associated domains (TADs). Hematopoietic-specific NPM1 knock-in alters TAD topology leading to disrupted regulation of the cell cycle as well as aberrant chromatin accessibility and homeotic gene expression, which results in myeloid differentiation block. Restoration of NPM1 within the nucleus re-establishes differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators that switch the oncogenic MIZ1/MYC regulatory axis in favor of interacting with coactivator NPM1/p300, and prevents NPM1-driven leukemogenesis. In sum, our data reveal that NPM1 reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation.

NPM1 mutation reprograms leukemic transcription network via reshaping TAD topology.

Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. PCa that relapses after hormonal therapies, referred to as castration resistant PCa (CRPC), often presents with metastases (mCRPC) that are the major cause of mortality. The few available therapies for mCRPC patients include taxanes docetaxel (DTX) and cabazitaxel (CBZ). However, development of resistance limits their clinical use. Mechanistically, resistance arises through upregulation of multidrug resistance (MDR) proteins such as MDR1/ABCB1, making ABCB1 an attractive therapeutic target. Yet, ABCB1 inhibitors failed to be clinically useful due to low specificity and toxicity issues. To study taxanes resistance, we produced CBZ resistant C4-2B cells (RC4-2B) and documented resistance to both CBZ and DTX in cell culture and in 3D prostaspheres settings. RNAseq identified increased expression of ABCB1 in RC4-2B, that was confirmed by immunoblotting and immunofluorescent analysis. ABCB1-specific inhibitor elacridar reversed CBZ and DTX resistance in RC4-2B cells, confirming ABCB1-mediated resistance mechanism. In a cell-based screen using a curated library of cytotoxic drugs, we found that DNA damaging compounds Camptothecin (CPT) and Cytarabine (Ara-C) overcame resistance as seen by similar cytotoxicity in parental C4-2B and resistant RC4-2B. Further, these compounds were cytotoxic to multiple PC cells resistant to taxanes with high ABCB1 expression and, therefore, can be used to conquer the acquired resistance to taxanes in PCa. Finally, inhibition of cyclin-dependent kinases 4/6 (CDK4/6) with small molecule inhibitors (CDK4/6i) potentiated cytotoxic effect of CPT or Ara-C in both parental and resistant cells. Overall, our findings indicate that DNA damaging agents CPT and Ara-C alone or in combination with CDK4/6i can be suggested as a new treatment regimen in CRPC patients, including those that are resistant to taxanes.

Overcoming ABCB1 mediated multidrug resistance in castration resistant prostate cancer.

Solitary fibrous tumors have gene expression signatures similar to those of neuroendocrine tumors.

Article	Year
FQI1: a transcription-methylation switch for cancer. Oncotarget\| PubMed ID: 28177910	2017
Loss of Dnmt3a Immortalizes Hematopoietic Stem Cells In Vivo. Cell reports\| PubMed ID: 29617651	2018
DNMT3A alterations associated with myeloid malignancies dictate differential responses to hypomethylating agents. Leukemia research\| PubMed ID: 32445940	2020
Alterations to in Hematologic Malignancies. Cancer research\| PubMed ID: 33087320	2021
HOXBLINC long non-coding RNA activation promotes leukemogenesis in NPM1-mutant acute myeloid leukemia. Nature communications\| PubMed ID: 33782403	2021
DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 34716195	2022
Combination strategies to promote sensitivity to cytarabine-induced replication stress in acute myeloid leukemia with and without DNMT3A mutations. Experimental hematology\| PubMed ID: 35306047	2022
The Impact of Inflammation-Induced Tumor Plasticity during Myeloid Transformation. Cancer discovery\| PubMed ID: 35924979	2022
miR-196b-TLR7/8 Signaling Axis Regulates Innate Immune Signaling and Myeloid Maturation in DNMT3A-Mutant AML. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 35943291	2022
NPM1 mutation reprograms leukemic transcription network via reshaping TAD topology. Leukemia\| PubMed ID: 37365294	2023
Overcoming ABCB1 mediated multidrug resistance in castration resistant prostate cancer. Cell death & disease\| PubMed ID: 39090086	2024
Decoding the identity of rare tumors. eLife\| PubMed ID: 39370970	2024

Olga A. Guryanova

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