Understanding the heterogeneous genetic mechanisms of tumor initiation in lymphoid leukemias (LL) will lead to improvements in prognostic classification and treatment regimens. In previous studies of mouse leukemias, we showed that retroviral insertion at the Evi32 locus leads to increased expression of Prdm14, a pluripotency gene implicated in the self-renewal capacity of embryonic stem cells and the early stages of breast cancer. Here we show that PRDM14 is also overexpressed in ~25% of human lymphoid neoplasms, with increased frequencies in T-cell acute LL (T-ALL) and hyperdiploid precursor B-cell acute LL (pre-B ALL). To test if Prdm14 overexpression could initiate leukemia, mice were transduced with bone marrow (BM) cells transfected with a Prdm14 expression vector. Lymphoid leukemias developed in 96% of female mice and 42% of male mice. Prior to the onset of leukemia, differentiation of transduced cells was biased up to 1000-fold towards cells with features of common lymphoid progenitors (CLP), and lymphoid differentiation showed a relative block at the pro-B stage. Microarray gene expression analysis of expanded CLP-like cells prior to the onset of leukemia demonstrated upregulation of genes involved in pluripotency, tumor initiation, early B-lineage commitment, Wnt/Ras signaling, and the epithelial-to-mesenchymal transition. Among the dysregulated genes were imprinted genes and non-coding RNAs including Dlk1 and Meg3, which are also key pluripotency mediators. Heightened expression of the estrogen-dependent oncogene, Myb, in tumors suggests a basis for the increased frequency of cancer in female mice. These data provide the first direct evidence for the association of Prdm14 with cancer initiation in an in vivo mouse model and in human lymphoid malignancies, while suggesting mechanisms for Prdm14’s mode of action.

SUMMARYPRDM14 functions in embryonic stem cell (ESC) maintenance to promote the expression of pluripotency-associated genes while suppressing differentiation genes. Expression of PRDM14 is tightly regulated and typically limited to ESCs and primordial germ cells; however, aberrant expression is associated with tumor initiation in a wide variety of human cancers, including breast cancer and leukemia. Here, we describe the generation of a Cre-recombinase-inducible mouse model for the spatial and temporal control of Prdm14 misexpression [ROSA26 floxed-stop Prdm14 (R26PR)]. When R26PR is mated to either of two Cre lines, Mx1-cre or MMTV-cre, mice develop early-onset T-cell acute lymphoblastic leukemia (T-ALL) with median overall survival of 41 and 64 days for R26PR;Mx1-cre and R26PR;MMTV-cre, respectively. T-ALL is characterized by the accumulation of immature single-positive CD8 cells and their widespread infiltration. Leukemia is preceded by a dramatic expansion of cells resembling hematopoietic stem cells and lymphoid-committed progenitors prior to disease onset, accompanied by a blockage in B-cell differentiation at the early pro-B stage. Rapid-onset PRDM14-induced T-ALL requires factors that are present in stem and progenitor cells: R26PR;dLck-cre animals, which express Prdm14 starting at the double-positive stage of thymocyte development, do not develop disease. PRDM14-induced leukemic cells contain high levels of activated NOTCH1 and downstream NOTCH1 targets, including MYC and HES1, and are sensitive to pharmacological inhibition of NOTCH1 with the γ-secretase inhibitor DAPT. Greater than 50% of human T-ALLs harbor activating mutations in NOTCH1; thus, our model carries clinically relevant molecular aberrations. The penetrance, short latency and involvement of the NOTCH1 pathway will make this hematopoietic R26PR mouse model ideal for future studies on disease initiation, relapse and novel therapeutic drug combinations. Furthermore, breeding R26PR to additional Cre lines will allow for the continued development of novel cancer models.

The TGF-β-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-β pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.

FOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with the neonatally lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in any human disease conditions. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking-in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 embryonic day (E)15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage-sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV.

PRDM14 is an epigenetic regulator known for maintaining embryonic stem cell identity and resetting potency in primordial germ cells. However, hematopoietic expression of Prdm14 at supraphysiological levels results in fully penetrant and rapid-onset T-cell acute lymphoblastic leukemia (T-ALL) in the mouse. Here, we show that PRDM14-induced T-ALLs are driven by NOTCH1, a frequently mutated driver of human T-ALL. Notch1 is activated in this murine model via RAG-dependent promoter deletions and subsequent production of truncated, ligand-independent protein from downstream regions of the Notch1 locus. These T-ALLs also have focal changes in H3K4me3 deposition at the Notch1 locus and global increases in both H3K4me1 and H3K4me3. Using a PRDM14-FLAG mouse model, we show that PRDM14 binds within an intron of Notch1 prior to leukemia development. Our data support the idea that PRDM14 binding promotes a chromatin state that allows access of the RAG recombinase complex to cryptic RAG signal sequences embedded at the Notch1 locus. Indeed, breeding into a RAG recombination-deficient background abrogates T-ALL development and prevents Notch1 deletions, while allowing for transient hematopoietic stem cell (HSC)-like pre-leukemia cell expansion. Together, our data suggest that PRDM14 expands a progenitor cell population while promoting a permissive epigenetic state for the creation of driver mutations (here, in Notch1), enabling cancer development through the misappropriation of endogenous cellular DNA recombination machinery.

Chloride intracellular channel 4 (CLIC4) is a tumor suppressor implicated in processes including growth arrest, differentiation, and apoptosis. CLIC4 protein expression is diminished in the tumor parenchyma during progression in squamous cell carcinoma (SCC) and other neoplasms, but the underlying mechanisms have not been identified. Data from The Cancer Genome Atlas suggest this is not driven by genomic alterations. However, screening and functional assays identified miR-142-3p as a regulator of CLIC4. CLIC4 and miR-142-3p expression are inversely correlated in head and neck (HN) SCC and cervical SCC, particularly in advanced stage cancers. In situ localization revealed that stromal immune cells, not tumor cells, are the predominant source of miR-142-3p in HNSCC. Furthermore, HNSCC single-cell expression data demonstrated that CLIC4 is lower in tumor epithelial cells than in stromal fibroblasts and endothelial cells. Tumor-specific downregulation of CLIC4 was confirmed in an SCC xenograft model concurrent with immune cell infiltration and miR-142-3p upregulation. These findings provide the first evidence of CLIC4 regulation by miRNA. Furthermore, the distinct localization of CLIC4 and miR-142-3p within the HNSCC tumor milieu highlight the limitations of bulk tumor analysis and provide critical considerations for both future mechanistic studies and use of miR-142-3p as a HNSCC biomarker.

Aberrant expression and activation of oncogenes in somatic cells has been associated with cancer initiation. Required for reacquisition of pluripotency in the developing germ cell, PRDM14 initiates lymphoblastic leukemia when misexpressed in murine bone marrow. Activation of pluripotency in somatic cells can lead to aneuploidy and copy number alterations during iPS cell generation, and we hypothesized that PRDM14-induced lymphoblastic leukemias would demonstrate significant chromosomal damage. High-resolution oligo array comparative genomic hybridization demonstrated infrequent aneuploidy but frequent amplification and deletion, with amplifications occurring in a 5:1 ratio with deletions. Many deletions (i.e., Cdkn2a, Ebf1, Pax5, Ikzf1) involved B-cell development genes and tumor suppressor genes, recapitulating deletions occurring in human leukemia. Pathways opposing senescence were frequently deactivated via Cdkn2a deletion or Tbx2 amplification, with corollary gene expression. Additionally, gene expression studies of abnormal pre-leukemic B-precursors showed downregulation of genes involved in chromosomal stability (i.e., Xrcc6) and failure to upregulate DNA repair pathways. We propose a model of leukemogenesis, triggered by pluripotency genes like Prdm14, which involves ongoing DNA damage and failure to activate non-homologous end-joining secondary to aberrant gene expression.

Cre-inducible mouse models are often utilized for the spatial and temporal expression of oncogenes. With the wide number of Cre recombinase lines available, inducible transgenesis represents a tractable approach to achieve discrete oncogene expression. Here, we describe a protocol for targeting Cre-inducible genes using a loxP-STOP-loxP approach to the ubiquitously expressed ROSA26 locus. Gene targeting provides several advantages over standard transgenic techniques, including a known site of integration and previously characterized pattern of expression. Historically, an inherent instability of ROSA26 targeting vectors has hampered the efficiency of developing ROSA26 knock-in lines. In this protocol, we provide individual steps for utilizing Gateway recombination for cloning, and detailed instructions for screening targeted ES cell clones. By following this protocol, one can achieve germline transmission of a ROSA26 knock-in line within several months.