Mediation of Ferroptosis Suppressor Protein 1 Expression via 4-Hydroxy-2-Nonenal Accumulation Contributes to Acquisition of Resistance to Apoptosis and Ferroptosis in Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. New therapeutic strategies are needed for the treatment of refractory DLBCL. 4-Hydroxy-2-nonenal (4-HNE) is a cytotoxic lipid peroxidation marker, which alters intracellular signaling and induces genetic mutations. Lipid peroxidation is associated with nonapoptotic cell death, called ferroptosis. However, the relationship between 4-HNE accumulation and feroptotic regulators in DLBCL has not been fully evaluated. Here, we aimed to evaluate the accumulation of lipid peroxide and the expression of ferroptosis suppressor protein 1 (FSP1) in DLBCL using immunohistochemistry. We found a significant increase in the expression of FSP1 in cases with nuclear 4-HNE accumulation (P = .021). Both nuclear and cytoplasmic 4-HNE accumulation and FSP1 positivity were independent predictors of worse prognosis. In vitro exposure to 4-HNE resulted in its concentration- and time-dependent intracellular accumulation and increased expression of FSP1. Furthermore, short-term (0.25 and 1.0 µM) or long-term (0.25 µM) exposure to 4-HNE induced resistance to not only apoptosis but also ferroptosis. Taken together, regulation of FSP1 through 4-HNE accumulation may attenuate resistance to cell death in treatment-resistant DLBCL and might help develop novel therapeutic strategies for refractory DLBCL.

Regulation of 4-HNE via SMARCA4 Is Associated with Worse Clinical Outcomes in Hepatocellular Carcinoma.

Accumulation of 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation, has various favorable and unfavorable effects on cancer cells; however, the clinicopathological significance of its accumulation in hepatocellular carcinoma (HCC) and its metabolic pathway remain unknown. This study analyzed 4-HNE accumulation and its clinicopathological significance in HCC. Of the 221 cases, 160 showed relatively low accumulation of 4-HNE in HCC tissues, which was an independent prognostic predictor. No correlation was found between 4-HNE accumulation and the expression of the antioxidant enzymes glutathione peroxidase 4, ferroptosis suppressor protein 1, and guanosine triphosphate cyclohydrolase 1. Therefore, we hypothesized that 4-HNE metabolism is up-regulated in HCC. A database search was focused on the transcriptional regulation of aldo-keto reductases, alcohol dehydrogenases, and glutathione-S-transferases, which are the metabolic enzymes of 4-HNE, and seven candidate transcription factor genes were selected. Among the candidate genes, the knockdown of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) increased 4-HNE accumulation. Immunohistochemical analysis revealed an inverse correlation between 4-HNE accumulation and SMARCA4 expression. These results suggest that SMARCA4 regulates 4-HNE metabolism in HCC. Therefore, targeting SMARCA4 provides a basis for a new therapeutic strategy for HCC via 4-HNE accumulation and increased cytotoxicity.

Artificial intelligence for evaluating the risk of gastric cancer: reliable detection and scoring of intestinal metaplasia with deep learning algorithms.

BACKGROUND AND AIMS: Gastric cancer (GC) is associated with chronic gastritis. To evaluate the risk, the Operative Link on Gastric Intestinal Metaplasia Assessment (OLGIM) system was constructed and showed a higher GC risk in stage III or IV patients, determined by the degree of intestinal metaplasia (IM). Although the OLGIM system is useful, evaluating the degree of IM requires substantial experience to produce precise scoring. Whole-slide imaging is becoming routine, but most artificial intelligence (AI) systems in pathology are focused on neoplastic lesions. METHODS: Hematoxylin and eosin-stained slides were scanned. Images were divided into each gastric biopsy tissue sample and labeled with an IM score. IM was scored as follows: 0 (no IM), 1 (mild IM), 2 (moderate IM), and 3 (severe IM). Overall, 5753 images were prepared. A deep convolutional neural network (DCNN) model, ResNet50, was used for classification. RESULTS: ResNet50 classified images with and without IM with a sensitivity of 97.7% and specificity of 94.6%. IM scores 2 and 3, involved as criteria of stage III or IV in the OLGIM system, were classified by ResNet50 in 18%. The respective sensitivity and specificity values of classifying IM between scores 0 and 1 and 2 and 3 were 98.5% and 94.9%, respectively. The IM scores classified by pathologists and the AI system were different in only 438 images (7.6%), and we found that ResNet50 tended to miss small foci of IM but successfully identified minimal IM areas that pathologists missed during the review. CONCLUSIONS: Our findings suggested that this AI system would contribute to evaluating the risk of GC accuracy, reliability, and repeatability with worldwide standardization.

Indirect CRISPR screening with photoconversion revealed key factors of drug resistance with cell-cell interactions.

Comprehensive screenings to clarify indirect cell-cell interactions, such as those in the tumor microenvironment, especially comprehensive assessments of supporting cells' effects, are challenging. Therefore, in this study, indirect CRISPR screening for drug resistance with cell-cell interactions was invented. The photoconvertible fluorescent protein Dendra2 was inducted to supporting cells and explored the drug resistance responsible factors of supporting cells with CRISPR screenings. Random mutated supporting cells co-cultured with leukemic cells induced drug resistance with cell-cell interactions. Supporting cells responsible for drug resistance were isolated with green-to-red photoconversion, and 39 candidate genes were identified. Knocking out C9orf89, MAGI2, MLPH, or RHBDD2 in supporting cells reduced the ratio of apoptosis of cancer cells. In addition, the low expression of RHBDD2 in supporting cells, specifically fibroblasts, of clinical pancreatic cancer showed a shortened prognosis, and a negative correlation with CXCL12 was observed. Indirect CRISPR screening was established to isolate the responsible elements of cell-cell interactions. This screening method could reveal unknown mechanisms in all kinds of cell-cell interactions by revealing live phenotype-inducible cells, and it could be a platform for discovering new targets of drugs for conventional chemotherapies.

Identification of NRAS Downstream Genes with CRISPR Activation Screening.

Mutations in NRAS constitutively activate cell proliferation signaling in malignant neoplasms, such as leukemia and melanoma, and the clarification of comprehensive downstream genes of NRAS might lead to the control of cell-proliferative signals of NRAS-driven cancers. We previously established that NRAS expression and proliferative activity can be controlled with doxycycline and named as THP-1 B11. Using a CRISPR activation library on THP-1 B11 cells with the NRAS-off state, survival clones were harvested, and 21 candidate genes were identified. By inducting each candidate guide RNA with the CRISPR activation system, DOHH, HIST1H2AC, KRT32, and TAF6 showed higher cell-proliferative activity. The expression of DOHH, HIST1H2AC, and TAF6 was definitely upregulated with NRAS expression. Furthermore, MEK inhibitors resulted in the decreased expression of DOHH, HIST1H2AC, and TAF6 proteins in parental THP-1 cells. The knockdown of DOHH, HIST1H2AC, and TAF6 was found to reduce proliferation in THP-1 cells, indicating that they are involved in the downstream proliferation of NRAS. These molecules are expected to be new therapeutic targets for NRAS-mutant leukemia cells.

Spatial and Quantitative Analysis of Tumor-Associated Macrophages: Intratumoral CD163-/PD-L1+ TAMs as a Marker of Favorable Clinical Outcomes in Triple-Negative Breast Cancer.

Tumor-associated macrophages (TAMs) and abnormalities in cancer cells affect cancer progression and response to therapy. TAMs are a major component of the tumor microenvironment (TME) in breast cancer, with their invasion affecting clinical outcomes. Programmed death-ligand 1 (PD-L1), a target of immune checkpoint inhibitors, acts as a suppressive signal for the surrounding immune system; however, its expression and effect on TAMs and the clinical outcome in breast cancer are unknown. In this study, we used high-throughput multiple immunohistochemistry to spatially and quantitatively analyze TAMs. We subjected 81 breast cancer specimens to immunostaining for CD68, CD163, PD-1, PD-L1, CD20, and pan-CK. In both stromal and intratumoral areas, the triple-negative subtype had significantly more CD68/CD163, CD68/PD-L1, and CD163/PD-L1 double-positive cells than the estrogen receptor (ER)/progesterone receptor (PR) subtype. Interestingly, a higher number of CD68+/PD-L1+/CK-/CD163- TAMs in the intratumoral area was correlated with a favorable recurrence rate (p = 0.048). These findings indicated that the specific subpopulation and localization of TAMs in the TME affect clinical outcomes in breast cancer.

Identification of the Factor That Leads Human Mesenchymal Stem Cell Lines into Decellularized Bone.

Hematopoiesis is maintained by the interaction of hematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (MSCs) in bone marrow microenvironments, called niches. Certain genetic mutations in MSCs, not HSCs, provoke some hematopoietic neoplasms, such as myelodysplastic syndrome. An in vivo bone marrow niche model using human MSC cell lines with specific genetic mutations and bone scaffolds is necessary to elucidate these interactions and the disease onset. We focused on decellularized bone (DCB) as a useful bone scaffold and attempted to induce human MSCs (UE7T-9 cells) into the DCB. Using the CRISPR activation library, we identified SHC4 upregulation as a candidate factor, with the SHC4 overexpression in UE7T-9 cells activating their migratory ability and upregulating genes to promote hematopoietic cell migration. This is the first study to apply the CRISPR library to engraft cells into decellularized biomaterials. SHC4 overexpression is essential for engrafting UE7T-9 cells into DCB, and it might be the first step toward creating an in vivo human-mouse hybrid bone marrow niche model.

Development and multi-institutional validation of an artificial intelligence-based diagnostic system for gastric biopsy.

To overcome the increasing burden on pathologists in diagnosing gastric biopsies, we developed an artificial intelligence-based system for the pathological diagnosis of gastric biopsies (AI-G), which is expected to work well in daily clinical practice in multiple institutes. The multistage semantic segmentation for pathology (MSP) method utilizes the distribution of feature values extracted from patches of whole-slide images (WSI) like pathologists' "low-power view" information of microscopy. The training dataset included WSIs of 4511 gastric biopsy tissues from 984 patients. In tissue-level validation, MSP AI-G showed better accuracy (91.0%) than that of conventional patch-based AI-G (PB AI-G) (89.8%). Importantly, MSP AI-G unanimously achieved higher accuracy rates (0.946 ± 0.023) than PB AI-G (0.861 ± 0.078) in tissue-level analysis, when applied to the cohorts of 10 different institutes (3450 samples of 1772 patients in all institutes, 198-555 samples of 143-206 patients in each institute). MSP AI-G had high diagnostic accuracy and robustness in multi-institutions. When pathologists selectively review specimens in which pathologist's diagnosis and AI prediction are discordant, the requirement of a secondary review process is significantly less compared with reviewing all specimens by another pathologist.

Quantitative high-throughput analysis of tumor infiltrating lymphocytes in breast cancer.

In breast cancer (BC), the development of cancer immunotherapy including immune checkpoint inhibitors has progressed. Tumor infiltrating lymphocytes (TILs) is one of the important factors for an immune response between tumor cells and immune cells in the tumor microenvironment, and the presence of TILs has been identified as predictors of response to chemotherapy. However, because complex mechanisms underlies the crosstalk between immune cells and cancer cells, the relationship between immune profiles in the tumor microenvironment and the efficacy of the immune checkpoint blocked has been unclear. Moreover, in many cases of breast cancer, the quantitative analysis of TILs and immuno-modification markers in a single tissue section are not studied. Therefore, we quantified detailed subsets of tumor infiltrating lymphocytes (TILs) from BC tissues and compared among BC subtypes. The TILs of BC tissues from 86 patients were classified using multiplex immunohistochemistry and an artificial intelligence-based analysis system based on T-cell subset markers, immunomodification markers, and the localization of TILs. The levels of CD4/PD1 and CD8/PD1 double-positive stromal TILs were significantly lower in the HER2- BC subtype (p <0.01 and p <0.05, respectively). In triple-negative breast cancer (TNBC), single marker-positive intratumoral TILs did not affect prognosis, however CD4/PDL1, CD8/PD1, and CD8/PDL1 double-positive TILs were significantly associated with TNBC recurrence (p<0.05, p<0.01, and p<0.001, respectively). TIL profiles differed among different BC subtypes, suggesting that the localization of TILs and their tumor-specific subsets influence the BC microenvironment.

Efficient Identification of the MYC Regulator with the Use of the CRISPR Library and Context-Matched Database Screenings.

MYC is a major oncogene that plays an important role in cell proliferation in human cancers. Therefore, the mechanism behind MYC regulation is a viable therapeutic target for the treatment of cancer. Comprehensive and efficient screening of MYC regulators is needed, and we had previously established a promoter screening system using fluorescent proteins and the CRISPR library. For the efficient identification of candidate genes, a database was used, for which mRNA expression was correlated with MYC using datasets featuring "Similar" and "Not exactly similar" contexts. INTS14 and ERI2 were identified using datasets featuring the "Similar" context group, and INTS14 and ERI2 were capable of enhancing MYC promoter activity. In further database analysis of human cancers, a higher expression of MYC mRNA was observed in the INTS14 mRNA high-expressing prostate and liver cancers. The knockdown of INTS14 in prostate cell lines resulted in decreased MYC mRNA and protein expression and also induced G0/1 arrest. This study confirmed that CRISPR screening combined with context-matched database screening is effective in identifying genes that regulate the MYC promoter. This method can be applied to other genes and is expected to be useful in identifying the regulators of other proto-oncogenes.

Proliferation and Self-Renewal Are Differentially Sensitive to NRASG12V Oncogene Levels in an Acute Myeloid Leukemia Cell Line.

NRAS proteins are central regulators of proliferation, survival, and self-renewal in leukemia. Previous work demonstrated that the effects of oncogenic NRAS in mediating proliferation and self-renewal are mutually exclusive within leukemia subpopulations and that levels of oncogenic NRAS vary between highly proliferative and self-renewing leukemia subpopulations. These findings suggest that NRAS activity levels may be important determinants of leukemic behavior. To define how oncogenic NRAS levels affect these functions, we genetically engineered an acute myeloid leukemia (AML) cell line, THP-1, to express variable levels of NRASG12V. We replaced the endogenous NRASG12D gene with a tetracycline-inducible and dose-responsive NRASG12V transgene. Cells lacking NRASG12V oncoprotein were cell-cycle arrested. Intermediate levels of NRASG12V induced maximal proliferation; higher levels led to attenuated proliferation, increased G1 arrest, senescence markers, and maximal self-renewal capacity. Higher levels of the oncoprotein also induced self-renewal and mitochondrial genes. We used mass cytometry (CyTOF) to define the downstream signaling events that mediate these differential effects. Not surprisingly, we found that the levels of such canonical RAS-effectors as pERK and p4EBP1 correlated with NRASG12V levels. ß-Catenin, a mediator of self-renewal, also correlated with NRASG12V levels. These signaling intermediates may mediate the differential effects of NRASG12V in leukemia biology. Together, these data reveal that oncogenic NRAS levels are important determinants of leukemic behavior explaining heterogeneity in phenotypes within a clone. This system provides a new model to study RAS oncogene addiction and RAS-induced self-renewal in AML. IMPLICATIONS: Different levels of activated NRAS may exert distinct effects on proliferation and self-renewal.

Stratification of lung squamous cell carcinoma based on ferroptosis regulators: Potential for new therapeutic strategies involving ferroptosis induction.

OBJECTIVES: Lung squamous cell carcinoma (LSCC) exhibits poor response to treatment compared with other lung cancer subtypes, resulting in worse prognosis. Therefore, new therapeutic strategies are required for advanced LSCC. Ferroptosis is a recently discovered nonapoptotic cell death caused by intracellular lipid peroxidation that can bring about effective cell death in cancer cells resistant to apoptosis. Hence, ferroptosis is a potential therapeutic strategy for refractory cancer. MATERIALS AND METHODS: In this study, we performed clinicopathological and molecular analyses on tumor specimens from 270 patients with squamous cell lung cancer, focusing on the expression of glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1), which are known to be key regulators of ferroptosis, and the accumulation of 4-hydroxynoneral (4-HNE), a lipid peroxidation marker. RESULTS: Immunohistochemistry revealed that patients with low 4-HNE accumulation and low levels of GPX4 or FSP1 had significantly worse prognoses than other patients (P = 0.001). This stratification was an independent prognostic predictor (P = 0.003). A dramatic cell death synergistic effect was observed on LSCC-derived LK-2 and EBC1 cells treated with GPX4 and FSP1 inhibitors. This effect was completely inhibited by treatment with the ferroptosis inhibitor. Notably, this was not the case in LK-2 cells treated with the apoptosis inhibitor, and in these cells, ferroptosis was induced. CONCLUSION: Ferroptosis regulators GPX4 and FSP1 are associated with lung squamous cell cancer cancer's prognosis. We present the clinicopathological and molecular basis of novel therapeutic strategies for refractory LSCC.

Pepsinogen I- and H+/K+-ATPase-immunohistochemical Positivity in Endoscopically Resected Early Gastric Neoplasia.

Gastric adenocarcinoma of the fundic gland type (GAFG) has been recently classified by the World Health Organization (WHO), however, clinicopathologic features of pepsinogen I- or H+/K+-ATPase-positive gastric tumors remain unclear. Therefore, this study evaluates the frequency and clinicopathologic features of those tumors, using a tissue microarray block to identify pepsinogen I- or H+/K+-ATPase-positive tumors from 810 endoscopically resected, early gastric epithelial tumors. The frequency of pepsinogen I-positive lesions was 2.1%, and that of H+/K+-ATPase-positive lesions was 2.0%. Pepsinogen I- or H+/K+-ATPase positivity was not observed in undifferentiated-type tumors, while gastric tumors with morphologic similarity to fundic glands were positive for pepsinogen I- or H+/K+-ATPase. We divided pepsinogen I- or H+/K+-ATPase-positive gastric tumors into group A, with fundic gland-like structure, or group B, without fundic gland-like structure. The frequency of group A was 1.6%: 46.2% were positive only for pepsinogen I and 53.8% for H+/K+-ATPase and pepsinogen I. The frequency of group B was 1.5%: 25% were positive only for pepsinogen I, 8.3% for H+/K+-ATPase and pepsinogen I, and 66.7% only for H+/K+-ATPase. The 2 tumor groups differed in location and endoscopic features. Hematoxylin and eosin staining showed that group B had more exposed tumors to the surface, larger nuclei, and more background atrophy than group A. Immunostaining showed significantly higher positivity rates for MUC5AC, CD10, CDX2, and p53 expression, and a higher Ki-67 labeling score. Our results provide novel insights into the pathology of early gastric tumors with histologic or immunohistochemical evidence of fundic gland differentiation.

HADHB, a fatty acid beta-oxidation enzyme, is a potential prognostic predictor in malignant lymphoma.

In haematological malignancies, such as malignant lymphoma, reprogramming of fatty acid metabolism favours tumour cell survival and drug resistance. Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), an enzyme involved in fatty acid beta-oxidation (FAO), is overexpressed in high-grade lymphoma and is a predictor of poor prognosis in diffuse large B-cell lymphoma (DLBCL). HADHB forms a heterodimer with HADHA and functions as an FAO enzyme together with HADHA; however, the relevance of its expression in malignant lymphoma is unknown. In this study, we investigated the roles and antitumour effects of HADHB expression in malignant lymphoma. Immunohistochemical analysis showed that HADHB was frequently overexpressed in the high-grade lymphoma subtype. HADHB overexpression was observed in 68% (87/128) of DLBCL cases and was an independent predictor of poor prognosis (p=0.001). In vitro analysis demonstrated that HADHB knockdown suppressed cell proliferation in LCL-K and MD901 cells (p<0.05). Additionally, treatment with the FAO inhibitor, ranolazine, increased cell death in control cells compared with that in HADHB knockdown LCL-K and MD901 cells (p<0.01). Cell death was also suppressed by the ferroptosis inhibitor, ferrosatin-1, in LCL-K and MD901 cells (p<0.05). Collectively, these findings provide basic evidence for the development of new cell death-based therapies for refractory malignant lymphoma. We plan to perform prospective studies and preclinical studies using animal models to confirm these results.

High prevalence of TERT aberrations in myxoid liposarcoma: TERT reactivation may play a crucial role in tumorigenesis.

Myxoid liposarcoma (MLPS) is genetically characterized by FUS-DDIT3 or EWSR1-DDIT3 gene fusion and the high frequency of hotspot mutations (C228T or C250T) in the promoter region of telomerase reverse transcriptase (TERT) that encodes the TERT protein. The latter leads to telomerase reactivation, a mechanism of telomere maintenance. Although the TERT promoter hotspot mutation is a poor prognostic factor in various tumors, its effect on MLPS has not been reported in detail. In the present study, we examined the clinicopathological characteristics, prognosis, and telomere maintenance mechanisms in 83 primary tumor samples of MLPS, which were resected surgically at the Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan, from 2008 to 2020. TERT promoter hotspot mutations were observed in 77% (63/82) cases, and alternative lengthening of telomeres (ALT) was absent in all cases. Among the cases without TERT promoter hotspot mutations, TERT rearrangements, and minor point mutations in the TERT promoter region were found in 3 and 2 cases, respectively. TERT mRNA expression was observed consistently even in patients for whom no genomic TERT aberrations were detected, and the presence of TERT promoter hotspot mutation did not correlate significantly with either overall and metastasis-free survival (P = .56, P = .83, respectively) or clinicopathological features. Therefore, patients with MLPS characteristically shows TERT expression and a high prevalence of TERT aberrations. Our findings suggest that TERT aberration is not prognostic factor, but might occur at an early stage and play a key role in tumorigenesis.

To Discover the Efficient and Novel Drug Targets in Human Cancers Using CRISPR/Cas Screening and Databases.

CRISPR/Cas has emerged as an excelle nt gene-editing technology and is used worldwide for research. The CRISPR library is an ideal tool for identifying essential genes and synthetic lethality targeted for cancer therapies in human cancers. Synthetic lethality is defined as multiple genetic abnormalities that, when present individually, do not affect function or survival, but when present together, are lethal. Recently, many CRISPR libraries are available, and the latest libraries are more accurate and can be applied to few cells. However, it is easier to efficiently search for cancer targets with their own screenings by effectively using databases of CRISPR screenings, such as Depmap portal, PICKLES (Pooled In-Vitro CRISPR Knockout Library Essentiality Screens), iCSDB, Project Score database, and CRISP-view. This review will suggest recent optimal CRISPR libraries and effective databases for Novel Approaches in the Discovery and Design of Targeted Therapies.

Current concept of stress fractures with an additional category of atypical fractures: a perspective review with representative images.

Stress fractures have traditionally been classified into three categories: fatigue fractures due to overuse of bone with normal elastic resistance; insufficiency fractures due to everyday physiological stress on fragile bone with poor elastic resistance; and pathologic fractures due to bone weakness involving tumors. The concept of atypical fractures has emerged and is considered a type of stress fracture. However, there has been some inconsistency in interpretation when using the traditional classification of stress fractures, and atypical femoral fractures (AFFs) can potentially be classified into subtypes: "typical" AFFs involving bone turnover suppression due to specific drugs (e.g. bisphosphonates) and fragility fractures of the bowed femoral shaft. In this article, the classification of stress fractures is redefined with the addition of atypical fractures as a fourth category, in which biological activity for fracture healing is absent, to promote consistent understanding and interpretation of clinical conditions involving stress fractures.

Characterization of mouse embryonic fibroblasts derived from Rassf6 knockout mice shows the implication of Rassf6 in the regulation of NF-κB signaling.

RASSF6 is a member of the tumor suppressor Ras association domain family (RASSF) proteins. We have reported using human cancer cell lines that RASSF6 induces apoptosis and cell cycle arrest via p53 and plays tumor suppressive roles. In this study, we generated Rassf6 knockout mice by CRISPR/Cas technology. Contrary to our expectation, Rassf6 knockout mice were apparently healthy. However, Rassf6-null mouse embryonic fibroblasts (MEF) were resistant against ultraviolet (UV)-induced apoptosis/cell cycle arrest and senescence. UV-induced p53-target gene expression was compromised, and DNA repair was delayed in Rassf6-null MEF. More importantly, KRAS active mutant promoted the colony formation of Rassf6-null MEF but not the wild-type MEF. RNA sequencing analysis showed that NF-κB signaling was enhanced in Rassf6-null MEF. Consistently, 7,12-dimethylbenz(a)anthracene (DMBA) induced skin inflammation in Rassf6 knockout mice more remarkably than in the wild-type mice. Hence, Rassf6 deficiency not only compromises p53 function but also enhances NF-κB signaling to lead to oncogenesis.

C/EBPß induces B-cell acute lymphoblastic leukemia and cooperates with BLNK mutations.

BLNK (BASH/SLP-65) encodes an adaptor protein that plays an important role in B-cell receptor (BCR) signaling. Loss-of-function mutations in this gene are observed in human pre-B acute lymphoblastic leukemia (ALL), and a subset of Blnk knock-out (KO) mice develop pre-B-ALL. To understand the molecular mechanism of the Blnk mutation-associated pre-B-ALL development, retroviral tagging was applied to KO mice using the Moloney murine leukemia virus (MoMLV). The Blnk mutation that significantly accelerated the onset of MoMLV-induced leukemia and increased the incidence of pre-B-ALL Cebpb was identified as a frequent site of retroviral integration, suggesting that its upregulation cooperates with Blnk mutations. Transgenic expression of the liver-enriched activator protein (LAP) isoform of Cebpb reduced the number of mature B-lymphocytes in the bone marrow and inhibited differentiation at the pre-BI stage. Furthermore, LAP expression significantly accelerated leukemogenesis in Blnk KO mice and alone acted as a B-cell oncogene. Furthermore, an inverse relationship between BLNK and C/EBPß expression was also noted in human pre-B-ALL cases, and the high level of CEBPB expression was associated with short survival periods in patients with BLNK-downregulated pre-B-ALL. These results indicate the association between the C/EBPß transcriptional network and BCR signaling in pre-B-ALL development and leukemogenesis. This study gives insight into ALL progression and suggests that the BCR/C/EBPß pathway can be a therapeutic target.