Cotargeting of BTK and MALT1 overcomes resistance to BTK inhibitors in mantle cell lymphoma.

Bruton's tyrosine kinase (BTK) is a proven target in mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma. However, resistance to BTK inhibitors is a major clinical challenge. We here report that MALT1 is one of the top overexpressed genes in ibrutinib-resistant MCL cells, while expression of CARD11, which is upstream of MALT1, is decreased. MALT1 genetic knockout or inhibition produced dramatic defects in MCL cell growth regardless of ibrutinib sensitivity. Conversely, CARD11-knockout cells showed antitumor effects only in ibrutinib-sensitive cells, suggesting that MALT1 overexpression could drive ibrutinib resistance via bypassing BTK/CARD11 signaling. Additionally, BTK knockdown and MALT1 knockout markedly impaired MCL tumor migration and dissemination, and MALT1 pharmacological inhibition decreased MCL cell viability, adhesion, and migration by suppressing NF-κB, PI3K/AKT/mTOR, and integrin signaling. Importantly, cotargeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in ibrutinib-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that MALT1 overexpression associates with resistance to BTK inhibitors in MCL, targeting abnormal MALT1 activity could be a promising therapeutic strategy to overcome BTK inhibitor resistance, and cotargeting of MALT1 and BTK should improve MCL treatment efficacy and durability as well as patient outcomes.

Genetic and epigenetic orchestration of Gfi1aa-Lsd1-cebpa in zebrafish neutrophil development.

Neutrophils are the most abundant vertebrate leukocytes and they are essential to host defense. Despite extensive investigation, the molecular network controlling neutrophil differentiation remains incompletely understood. GFI1 is associated with several myeloid disorders, but its role and the role of its co-regulators in granulopoiesis and pathogenesis are far from clear. Here, we demonstrate that zebrafish gfi1aa deficiency induces excessive neutrophil progenitor proliferation, accumulation of immature neutrophils from the embryonic stage, and some phenotypes similar to myelodysplasia syndrome in adulthood. Both genetic and epigenetic analyses demonstrate that immature neutrophil accumulation in gfi1aa-deficient mutants is due to upregulation of cebpa transcription. Increased transcription was associated with Lsd1-altered H3K4 methylation of the cebpa regulatory region. Taken together, our results demonstrate that Gfi1aa, Lsd1 and cebpa form a regulatory network that controls neutrophil development, providing a disease progression-traceable model for myelodysplasia syndrome. Use of this model could provide new insights into the molecular mechanisms underlying GFI1-related myeloid disorders as well as a means by which to develop targeted therapeutic approaches for treatment.

Longitudinal single-cell profiling reveals molecular heterogeneity and tumor-immune evolution in refractory mantle cell lymphoma.

The mechanisms driving therapeutic resistance and poor outcomes of mantle cell lymphoma (MCL) are incompletely understood. We characterize the cellular and molecular heterogeneity within and across patients and delineate the dynamic evolution of tumor and immune cell compartments at single cell resolution in longitudinal specimens from ibrutinib-sensitive patients and non-responders. Temporal activation of multiple cancer hallmark pathways and acquisition of 17q are observed in a refractory MCL. Multi-platform validation is performed at genomic and cellular levels in PDX models and larger patient cohorts. We demonstrate that due to 17q gain, BIRC5/survivin expression is upregulated in resistant MCL tumor cells and targeting BIRC5 results in marked tumor inhibition in preclinical models. In addition, we discover notable differences in the tumor microenvironment including progressive dampening of CD8+ T cells and aberrant cell-to-cell communication networks in refractory MCLs. This study reveals diverse and dynamic tumor and immune programs underlying therapy resistance in MCL.

Slc20a1b is essential for hematopoietic stem/progenitor cell expansion in zebrafish.

Hematopoietic stem and progenitor cells (HSPCs) are able to self-renew and can give rise to all blood lineages throughout their lifetime, yet the mechanisms regulating HSPC development have yet to be discovered. In this study, we characterized a hematopoiesis defective zebrafish mutant line named smu07, which was obtained from our previous forward genetic screening, and found the HSPC expansion deficiency in the mutant. Positional cloning identified that slc20a1b, which encodes a sodium phosphate cotransporter, contributed to the smu07 blood phenotype. Further analysis demonstrated that mutation of slc20a1b affects HSPC expansion through cell cycle arrest at G2/M phases in a cell-autonomous manner. Our study shows that slc20a1b is a vital regulator for HSPC proliferation in zebrafish early hematopoiesis and provides valuable insights into HSPC development.

Gfi1aa/Lsd1 Facilitates Hemangioblast Differentiation Into Primitive Erythrocytes by Targeting etv2 and sox7 in Zebrafish.

The first wave of hematopoiesis is the primitive hematopoiesis, which produces embryonic erythroid and myeloid cells. Primitive erythrocytes are thought to be generated from bipotent hemangioblasts, but the molecular basis remains unclear. Transcriptional repressors Gfi1aa and Gfi1b have been shown to cooperatively promote primitive erythrocytes differentiation from hemangioblasts in zebrafish. However, the mechanism of these repressors during the primitive wave is largely unknown. Herein, by functional analysis of zebrafish gfi1aa smu10 , gfi1b smu11 , gfi1ab smu12 single, double, and triple mutants, we found that Gfi1aa not only plays a predominant role in primitive erythropoiesis but also synergizes with Gfi1ab. To screen Gfi1aa downstream targets, we performed RNA-seq and ChIP-seq analysis and found two endothelial transcription factors, etv2 and sox7, to be repressed by Gfi1aa. Genetic analysis demonstrated Gfi1aa to promote hemangioblast differentiation into primitive erythrocytes by inhibiting both etv2 and sox7 in an Lsd1-dependent manner. Moreover, the H3K4me1 level of etv2 and sox7 were increased in gfi1aa mutant. Taken together, these results suggest that Gfi1aa/Lsd1-dependent etv2/sox7 downregulation is critical for hemangioblast differentiation during primitive hematopoiesis by inhibition of endothelial specification. The different and redundant roles for Gfi1(s), as well as their genetic and epigenetic regulation during primitive hematopoiesis, help us to better know the molecular basis of the primitive hematopoiesis and sheds light on the understanding the Gfi1(s) related pathogenesis.

Alas1 is essential for neutrophil maturation in zebrafish.

Neutrophils play essential roles in innate immunity and are the first responders to kill foreign micro-organisms, a function that partially depends on their granule content. The complicated regulatory network of neutrophil development and maturation remains largely unknown. Here we utilized neutrophil-deficient zebrafish to identify a novel role of Alas1, a heme biosynthesis pathway enzyme, in neutrophil development. We showed that Alas1-deficient zebrafish exhibited proper neutrophil initiation, but further neutrophil maturation was blocked due to heme deficiency, with lipid storage and granule formation deficiencies, and loss of heme-dependent granule protein activities. Consequently, Alas1-deficient zebrafish showed impaired bactericidal ability and augmented inflammatory responses when challenged with Escherichia coli These findings demonstrate the important role of Alas1 in regulating neutrophil maturation and physiological function through the heme. Our study provides an in vivo model of Alas1 deficiency and may be useful to evaluate the progression of heme-related disorders in order to facilitate the development of drugs and treatment strategies for these diseases.

miR-192, a prognostic indicator, targets the SLC39A6/SNAIL pathway to reduce tumor metastasis in human hepatocellular carcinoma.

Metastasis is one of the causes of cancer death. Functions and mechanisms of microRNAs (miRNAs) involved in hepatocellular carcinoma (HCC) metastasis are largely unknown. Here, a miRNA microarray analysis was performed in MHCC-97L, MHCC-97H and HCC-LM3 cells with gradually increasing metastatic potential to disclose crucial miRNAs involved in HCC metastasis. miR-192 expression decreased and negatively correlated with vascular invasion in HCC specimens. Gain and loss of function studies revealed that miR-192 significantly suppressed metastasis of HCC cells in vitro and in vivo. Solute carrier family 39 member 6 (SLC39A6) was identified as a direct and functional target of miR-192. In addition, SLC39A6 negatively correlated with miR-192 in HCC samples and promoted HCC cell migration and invasion. Moreover, miR-192 decreased SLC39A6 expression, subsequently downregulating SNAIL and upregulating E-cadherin expression. Suppression of migration and invasion caused by miR-192 overexpression was alleviated by exogenous Snail expression. Intriguingly, lower miR-192 expression and higher SLC39A6 expression significantly contributed to poorer outcomes in HCC patients. Multivariate analysis indicated that miR-192 was an independent and significant predictor of HCC patient overall survival. In conclusion, we newly determined that miR-192 targeted the SLC39A6/SNAIL pathway to reduce tumor metastasis in HCC cells. This axis provided insights into the mechanism underlying miRNA regulation of HCC metastasis and a novel therapeutic target for HCC treatment.

MicroRNA-127-5p targets the biliverdin reductase B/nuclear factor-κB pathway to suppress cell growth in hepatocellular carcinoma cells.

Nuclear factor-κB (NF-κB) activation is one of the major mediators of inflammation-induced cancer cell growth and progression. In previous studies, we screened a series of microRNAs (miRNAs) that targeted the NF-κB signaling pathway. In this study, we showed that miR-127-5p suppressed NF-κB activity through inhibition of p65 nuclear translocation. In addition, miR-127-5p also inhibited the transcription of downstream targets of the NF-κB signaling pathway. While exploring the mechanism of the inhibition of NF-κB activity by miR-127-5p, we found that miR-127-5p decreased the phosphorylation of p65. MicroRNA-127-5p inhibited the growth and colony formation of hepatocellular carcinoma (HCC) cells and decreased biliverdin reductase B (BLVRB) expression by directly binding to its 3'-UTR. RNA interference of BLVRB suppressed HCC cell growth, whereas the overexpression of BLVRB promoted HCC cell growth. Furthermore, BLVRB blockade inhibited the phosphorylation of p65 protein and the expression of downstream targets of the NF-κB signaling pathway, mimicking the function of miR-127-5p. The restoration of BLVRB in HCC cells overexpressing miR-127-5p impaired the suppression of HCC growth by miR-127-5p. Moreover, miR-127-5p was downregulated in 58% of HCC samples. In summary, we found that miR-127-5p suppressed NF-κB activity by directly targeting BLVRB in HCC cells, and this finding improves our understanding of the molecular mechanism of inflammation-induced HCC growth and proliferation and the successful inhibition of NF-κB activity by cancer treatment.

GNAI3 inhibits tumor cell migration and invasion and is post-transcriptionally regulated by miR-222 in hepatocellular carcinoma.

Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 (GNAI3) is involved in many biological processes. However, its biological function in hepatocellular carcinoma (HCC) remains unclear. An immunohistochemical staining analysis revealed that GNAI3 protein was down-regulated in HCC compared to non-cancerous liver. Furthermore, transwell assays indicated that GNAI3 inhibits HCC cell migration and invasion. Using software predictions and experimental screening, we found that miR-222 could directly bind to GNAI3 mRNA and decrease GNAI3 protein expression in HCC cells. Moreover, miR-222 was up-regulated in HCC and negatively correlated with GNAI3 protein expression. These results indicated that down-regulation of GNAI3 might be caused by up-regulation of miR-222 in HCC. In conclusion, GNAI3 was down-regulated by miR-222 in HCC, and this deregulation promoted migration and invasion of HCC. These findings extended our insight into the complex molecular mechanisms underlying the invasion and metastasis of HCC and may provide new therapeutic targets.

Genome-wide miRNA-profiling of aflatoxin B1-induced hepatic injury using deep sequencing.

Aflatoxin B1 is a potent carcinogen which can induce** hepatocellular carcinoma (HCC) in mammals. Though microRNAs are known to play important roles in tumorigenesis, the functional complexity of microRNAs in AFB1-induced hepatocellular tumorigenesis has not yet been elucidated. Here, we applied Illumina deep sequencing technology for high-throughput profiling of microRNA in rat liver tissue before and after treatment with aflatoxin B1. Analysis of mature miRNAs from different arms of pre-miRNAs allowed us to identify the predominant form of miRNA. We studied the differential expression profile of miRNAs in two libraries, identifying several cancer-related microRNAs which exhibit abnormal expression. KEGG analysis indicated that predicted target genes of differentially expressed miRNAs are involved in cancer-related pathways. Bioinformatic analysis predicted 16 potential novel miRNAs. Our work provides new insights at the miRNA level into AFB1-induced hepatic injury which may lead to HCC.

Screening for a single-chain variable-fragment antibody that can effectively neutralize the cytotoxicity of the Vibrio parahaemolyticus thermolabile hemolysin.

Vibrio parahaemolyticus is a halophilic bacterium that is widely distributed in water resources. The bacterium causes lethal food-borne diseases and poses a serious threat to human and animal health all over the world. The major pathogenic factor of V. parahaemolyticus is thermolabile hemolysin (TLH), encoded by the tlh gene, but its toxicity mechanisms are unknown. A high-affinity antibody that can neutralize TLH activity effectively is not available. In this study, we successfully expressed and purified the TLH antigen and discovered a high-affinity antibody to TLH, named scFv-LA3, by phage display screening. Cytotoxicity analysis showed that scFv-LA3 has strong neutralization effects on TLH-induced cell toxicity.

Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA-DNA hybridization on a microarray.

In this paper, we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains, using multiplex PCR and DNA-DNA hybridization. Multiplex PCR was used to simultaneously amplify three diagnostic genes (tlh, tdh and fla) that serve as molecular markers of V. parahaemolyticus. Biotinylated PCR products were hybridized to primers immobilized on a microarray, and detected by chemiluminesce with avidin-conjugated alkaline phosphatase. With this method, forty-five samples were tested. Eight known virulent strains (tlh(+)/tdh(+)/fla(+)) and four known avirulent strains (tlh(+)/tdh(-)/fla(+)) of the V. parahaemolyticus were successfully detected, and no non-specific hybridization and cross-hybridization reaction were found from fifteen closely-related strains (tlh(-)/tdh(-)/fla(+)) of the Vibrio spp. In addition, all the other eighteen strains of non-Vibrio bacteria (tlh(-)/tdh(-)/fla(-)) gave negative results. The DNA microarray successfully distinguished V. parahaemolyticus from other Vibrio spp. The results demonstrated that this was an efficient and robust method for identifying virulent strains of V. parahaemolyticus.