Cbl and Cbl-b ubiquitin ligases are essential for intestinal epithelial stem cell maintenance.

Receptor tyrosine kinases (RTKs) control stem cell maintenance vs. differentiation decisions. Casitas B-lineage lymphoma (CBL) family ubiquitin ligases are negative regulators of RTKs, but their stem cell regulatory roles remain unclear. Here, we show that Lgr5+ intestinal stem cell (ISC)-specific inducible Cbl-knockout (KO) on a Cblb null mouse background (iDKO) induced rapid loss of the Lgr5 Hi ISCs with transient expansion of the Lgr5 Lo transit-amplifying population. LacZ-based lineage tracing revealed increased ISC commitment toward enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro, Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single-cell RNA sequencing in organoids identified Akt-mTOR (mammalian target of rapamycin) pathway hyperactivation upon iDKO, and pharmacological Akt-mTOR axis inhibition rescued the iDKO defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine-tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.

Cbl and Cbl-b Ubiquitin Ligases are Essential for Intestinal Epithelial Stem Cell Maintenance.

Among the signaling pathways that control the stem cell self-renewal and maintenance vs. acquisition of differentiated cell fates, those mediated by receptor tyrosine kinase (RTK) activation are well established as key players. CBL family ubiquitin ligases are negative regulators of RTKs but their physiological roles in regulating stem cell behaviors are unclear. While hematopoietic Cbl/Cblb knockout (KO) leads to a myeloproliferative disease due to expansion and reduced quiescence of hematopoietic stem cells, mammary epithelial KO led to stunted mammary gland development due to mammary stem cell depletion. Here, we examined the impact of inducible Cbl/Cblb double-KO (iDKO) selectively in the Lgr5-defined intestinal stem cell (ISC) compartment. Cbl/Cblb iDKO led to rapid loss of the Lgr5 Hi ISC pool with a concomitant transient expansion of the Lgr5 Lo transit amplifying population. LacZ reporter-based lineage tracing showed increased ISC commitment to differentiation, with propensity towards enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro , Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single cell RNAseq analysis of organoids revealed Akt-mTOR pathway hyperactivation in iDKO ISCs and progeny cells, and pharmacological inhibition of the Akt-mTOR axis rescued the organoid maintenance and propagation defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.

Interpreting and de-noising genetically engineered barcodes in a DNA virus.

The concept of a nucleic acid barcode applied to pathogen genomes is easy to grasp and the many possible uses are straightforward. But implementation may not be easy, especially when growing through multiple generations or assaying the pathogen long-term. The potential problems include: the barcode might alter fitness, the barcode may accumulate mutations, and construction of the marked pathogens may result in unintended barcodes that are not as designed. Here, we generate approximately 5,000 randomized barcodes in the genome of the prototypic small DNA virus murine polyomavirus. We describe the challenges faced with interpreting the barcode sequences obtained from the library. Our Illumina NextSeq sequencing recalled much greater variation in barcode sequencing reads than the expected 5,000 barcodes-necessarily stemming from the Illumina library processing and sequencing error. Using data from defined control virus genomes cloned into plasmid backbones we develop a vetted post-sequencing method to cluster the erroneous reads around the true virus genome barcodes. These findings may foreshadow problems with randomized barcodes in other microbial systems and provide a useful approach for future work utilizing nucleic acid barcoded pathogens.

The histone deacetylase inhibitor M344 as a multifaceted therapy for pancreatic cancer.

The histone deacetylase (HDAC) inhibitor vorinostat, used with gemcitabine and other therapies, has been effective in treatment of experimental models of pancreatic cancer. In this study, we demonstrated that M344, an HDAC inhibitor, is efficacious against pancreatic cancer in vitro and in vivo, alone or with gemcitabine. By 24 hours post-treatment, M344 augments the population of pancreatic cancer cells in G1, and at a later time point (48 hours) it increases apoptosis. M344 inhibits histone H3 deacetylation and slows pancreatic cancer cell proliferation better than vorinostat, and it does not decrease the viability of a non-malignant cell line more than vorinostat. M344 also elevates pancreatic cancer cell major histocompatibility complex (MHC) class I molecule expression, potentially increasing the susceptibility of pancreatic cancer cells to T cell lysis. Taken together, our findings support further investigation of M344 as a pancreatic cancer treatment.

Nanoformulation of CCL21 greatly increases its effectiveness as an immunotherapy for neuroblastoma.

Neuroblastoma is the most commonly diagnosed extracranial solid tumor in children. The patients with aggressive metastatic disease or refractory/relapsed neuroblastoma currently face a dismally low chance of survival. Thus, there is a great need for more effective therapies for this illness. In previous studies, we, as well as others, showed that the immune cell chemoattractant C-C motif chemokine ligand 21 (CCL21) is effective as an intratumoral therapy able to slow the growth of cancers. In this current study, we developed and tested an injectable, slow-release, uniform, and optimally loaded alginate nanoformulation of CCL21 as a means to provide prolonged intratumoral treatment. The alginate-nanoformulated CCL21, when injected intratumorally into mice bearing neuroblastoma lesions, significantly prolonged survival and decreased the tumor growth rate compared to CCL21 alone, empty nanoparticles, or buffer. Notably, we also observed complete tumor clearance and subsequent full protection against tumor rechallenge in 33% of nanoformulated CCL21-treated mice. Greater intratumoral presence of nanoformulated CCL21, compared to free CCL21, at days 1 and 2 after treatment ended was confirmed through fluorescent labeling and tracking. Nanoformulated CCL21-treated tumors exhibited a general pattern of prolonged increases in anti-tumor cytokines and relatively lower levels of pro-tumor cytokines in comparison to tumors treated with CCL21 alone or buffer only. Thus, this novel nanoformulation of CCL21 is an effective treatment for neuroblastoma, and may have potential for the delivery of CCL21 to other types of solid tumors in the future and as a slow-release delivery modality for other immunotherapies.

HLA-B influences integrin beta-1 expression and pancreatic cancer cell migration.

Human leukocyte antigen (HLA) class I molecules present antigenic peptides to cytotoxic T cells, causing lysis of malignant cells. Transplantation biology studies have implicated HLA class I molecules in cell migration, but there has been little evidence presented that they influence cancer cell migration, a contributing factor in metastasis. In this study, we examined the effect of HLA-B on pancreatic cancer cell migration. HLA-B siRNA transfection increased the migration of the S2-013 pancreatic cancer cells but, in contrast, reduced migration of the PANC-1 and MIA PaCa-2 pancreatic cancer cell lines. Integrin molecules have previously been implicated in the upregulation of pancreatic cancer cell migration, and knockdown of HLA-B in S2-013 cells heightened the expression of integrin beta 1 (ITGB1), but in the PANC-1 and MIA PaCa-2 cells HLA-B knockdown diminished ITGB1 expression. A transmembrane sequence in an S2-013 HLA-B heavy chain matches a corresponding sequence in HLA-B in the BxPC-3 pancreatic cancer cell line, and knockdown of BxPC-3 HLA-B mimics the effect of S2-013 HLA-B knockdown on migration. In total, our findings indicate that HLA-B influences the expression of ITGB1 in pancreatic cancer cells, with concurrent distinctions in transmembrane sequences and effects on the migration of the cells.

EHD1 and RUSC2 Control Basal Epidermal Growth Factor Receptor Cell Surface Expression and Recycling.

Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.

Molecular profiling of single neurons of known identity in two ganglia from the crab Cancer borealis.

Understanding circuit organization depends on identification of cell types. Recent advances in transcriptional profiling methods have enabled classification of cell types by their gene expression. While exceptionally powerful and high throughput, the ground-truth validation of these methods is difficult: If cell type is unknown, how does one assess whether a given analysis accurately captures neuronal identity? To shed light on the capabilities and limitations of solely using transcriptional profiling for cell-type classification, we performed 2 forms of transcriptional profiling-RNA-seq and quantitative RT-PCR, in single, unambiguously identified neurons from 2 small crustacean neuronal networks: The stomatogastric and cardiac ganglia. We then combined our knowledge of cell type with unbiased clustering analyses and supervised machine learning to determine how accurately functionally defined neuron types can be classified by expression profile alone. The results demonstrate that expression profile is able to capture neuronal identity most accurately when combined with multimodal information that allows for post hoc grouping, so analysis can proceed from a supervised perspective. Solely unsupervised clustering can lead to misidentification and an inability to distinguish between 2 or more cell types. Therefore, this study supports the general utility of cell identification by transcriptional profiling, but adds a caution: It is difficult or impossible to know under what conditions transcriptional profiling alone is capable of assigning cell identity. Only by combining multiple modalities of information such as physiology, morphology, or innervation target can neuronal identity be unambiguously determined.

Beta 2-microglobulin regulates amyloid precursor-like protein 2 expression and the migration of pancreatic cancer cells.

Beta 2-microglobulin (ß2m) is a component of the major histocompatibility complex (MHC) class I molecule, which presents tumor antigens to T lymphocytes to trigger cancer cell destruction. Notably, ß2m has been reported as persistently expressed, rather than down regulated, in some tumor types. For renal cell and oral squamous cell carcinomas, ß2m expression has been linked to increased migratory capabilities. The migratory ability of pancreatic cancer cells contributes to their metastatic tendencies and lethal nature. Therefore, in this study, we examined the impact of ß2m on pancreatic cancer cell migration. We found that ß2m protein is amply expressed in several human pancreatic cancer cell lines (S2-013, PANC-1, and MIA PaCa-2). Reducing ß2m expression by short interfering RNA (siRNA) transfection significantly slowed the migration of the PANC-1 and S2-013 cancer cell lines, but increased the migration of the MIA PaCa-2 cell line. The amyloid precursor-like protein 2 (APLP2) has been documented as contributing to pancreatic cancer cell migration, invasiveness, and metastasis. We have previously shown that ß2m/HLA class I/peptide complexes associate with APLP2 in S2-013 cells, and in this study we also detected their association in PANC-1 cells but not MIA PaCa-2 cells. In addition, siRNA down regulation of ß2m expression diminished the expression of APLP2 in S2-013 and PANC-1 but heightened the level of APLP2 in MIA PaCa-2 cells, consistent with our migration data and co-immunoprecipitation data. Thus, our findings indicate that ß2m regulates pancreatic cancer cell migration, and furthermore suggest that APLP2 is an intermediary in this process.

Genomics of Clostridium taeniosporum, an organism which forms endospores with ribbon-like appendages.

Clostridium taeniosporum, a non-pathogenic anaerobe closely related to the C. botulinum Group II members, was isolated from Crimean lake silt about 60 years ago. Its endospores are surrounded by an encasement layer which forms a trunk at one spore pole to which about 12-14 large, ribbon-like appendages are attached. The genome consists of one 3,264,813 bp, circular chromosome (with 26.6% GC) and three plasmids. The chromosome contains 2,892 potential protein coding sequences: 2,124 have specific functions, 147 have general functions, 228 are conserved but without known function and 393 are hypothetical based on the fact that no statistically significant orthologs were found. The chromosome also contains 101 genes for stable RNAs, including 7 rRNA clusters. Over 84% of the protein coding sequences and 96% of the stable RNA coding regions are oriented in the same direction as replication. The three known appendage genes are located within a single cluster with five other genes, the protein products of which are closely related, in terms of sequence, to the known appendage proteins. The relatedness of the deduced protein products suggests that all or some of the closely related genes might code for minor appendage proteins or assembly factors. The appendage genes might be unique among the known clostridia; no statistically significant orthologs were found within other clostridial genomes for which sequence data are available. The C. taeniosporum chromosome contains two functional prophages, one Siphoviridae and one Myoviridae, and one defective prophage. Three plasmids of 5.9, 69.7 and 163.1 Kbp are present. These data are expected to contribute to future studies of developmental, structural and evolutionary biology and to potential industrial applications of this organism.

Role of the EHD Family of Endocytic Recycling Regulators for TCR Recycling and T Cell Function.

T cells use the endocytic pathway for key cell biological functions, including receptor turnover and maintenance of the immunological synapse. Some of the established players include the Rab GTPases, the SNARE complex proteins, and others, which function together with EPS-15 homology domain-containing (EHD) proteins in non-T cell systems. To date, the role of the EHD protein family in T cell function remains unexplored. We generated conditional EHD1/3/4 knockout mice using CD4-Cre and crossed these with mice bearing a myelin oligodendrocyte glycoprotein-specific TCR transgene. We found that CD4+ T cells from these mice exhibited reduced Ag-driven proliferation and IL-2 secretion in vitro. In vivo, these mice exhibited reduced severity of experimental autoimmune encephalomyelitis. Further analyses showed that recycling of the TCR-CD3 complex was impaired, leading to increased lysosomal targeting and reduced surface levels on CD4+ T cells of EHD1/3/4 knockout mice. Our studies reveal a novel role of the EHD family of endocytic recycling regulatory proteins in TCR-mediated T cell functions.

CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies.

Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b Importantly, primary human CBL mutated (CBLmut ) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies.

An essential role of CBL and CBL-B ubiquitin ligases in mammary stem cell maintenance.

The ubiquitin ligases CBL and CBL-B are negative regulators of tyrosine kinase signaling with established roles in the immune system. However, their physiological roles in epithelial tissues are unknown. Here, we used MMTV-Cre-mediated Cbl gene deletion on a Cbl-b null background, as well as a tamoxifen-inducible mammary stem cell (MaSC)-specific Cbl and Cbl-b double knockout (Cbl/Cbl-b DKO) using Lgr5-EGFP-IRES-CreERT2, to demonstrate a mammary epithelial cell-autonomous requirement of CBL and CBL-B in the maintenance of MaSCs. Using a newly engineered tamoxifen-inducible Cbl and Cbl-b deletion model with a dual fluorescent reporter (Cblflox/flox; Cbl-bflox/flox; Rosa26-CreERT; mT/mG), we show that Cbl/Cbl-b DKO in mammary organoids leads to hyperactivation of AKT-mTOR signaling with depletion of MaSCs. Chemical inhibition of AKT or mTOR rescued MaSCs from Cbl/Cbl-b DKO-induced depletion. Our studies reveal a novel, cell-autonomous requirement of CBL and CBL-B in epithelial stem cell maintenance during organ development and remodeling through modulation of mTOR signaling.

A novel CBL-Bflox/flox mouse model allows tissue-selective fully conditional CBL/CBL-B double-knockout: CD4-Cre mediated CBL/CBL-B deletion occurs in both T-cells and hematopoietic stem cells.

CBL-family ubiquitin ligases are critical negative regulators of tyrosine kinase signaling, with a clear redundancy between CBL and CBL-B evident in the immune cell and hematopoietic stem cell studies. Since CBL and CBL-B are negative regulators of immune cell activation, elimination of their function to boost immune cell activities could be beneficial in tumor immunotherapy. However, mutations of CBL are associated with human leukemias, pointing to tumor suppressor roles of CBL proteins; hence, it is critical to assess the tumor-intrinsic roles of CBL and CBL-B in cancers. This has not been possible since the only available whole-body CBL-B knockout mice exhibit constitutive tumor rejection. We engineered a new CBL-Bflox/flox mouse, combined this with an existing CBLflox/flox mouse to generate CBLflox/flox; CBL-Bflox/flox mice, and tested the tissue-specific concurrent deletion of CBL and CBL-B using the widely-used CD4-Cre transgenic allele to produce a T-cell-specific double knockout. Altered T-cell development, constitutive peripheral T-cell activation, and a lethal multi-organ immune infiltration phenotype largely resembling the previous Lck-Cre driven floxed-CBL deletion on a CBL-B knockout background establish the usefulness of the new model for tissue-specific CBL/CBL-B deletion. Unexpectedly, CD4-Cre-induced deletion in a small fraction of hematopoietic stem cells led to expansion of certain non-T-cell lineages, suggesting caution in the use of CD4-Cre for T-cell-restricted gene deletion. The establishment of a new model of concurrent tissue-selective CBL/CBL-B deletion should allow a clear assessment of the tumor-intrinsic roles of CBL/CBL-B in non-myeloid malignancies and help test the potential for CBL/CBL-B inactivation in immunotherapy of tumors.

Dynamic postnatal developmental and sex-specific neuroendocrine effects of prenatal polychlorinated biphenyls in rats.

Gestational exposures to estrogenic compounds, both endogenous hormones and exogenous endocrine-disrupting chemicals (EDCs), have long-term effects on reproductive physiology and behavior. We tested the hypothesis that prenatal treatment of rats with low doses of Aroclor 1221 (A1221), a weakly estrogenic polychlorinated biphenyl mix previously used in industry, or estradiol benzoate (EB), alters development of the hypothalamus in a sexually dimorphic manner and subsequently perturbs reproductive function. Pregnant Sprague-Dawley rats were injected on embryonic days 16 and 18 with vehicle (dimethylsulfoxide), A1221 (1 mg/kg), or EB (50 µg/kg). Developmental milestones were monitored, and on postnatal days 15, 30, 45, and 90, 1 male and 1 female per litter were euthanized. Because of their key roles in the mediation of steroid actions on reproductive function, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) were punched for a low-density quantitative PCR array of 48 neuroendocrine genes and analysis of DNA methylation of a subset of genes. Gestational exposure to A1221 or EB delayed the timing of puberty in males and disrupted estrous cyclicity in females. In the AVPV, 28 genes were affected by treatment in a developmental stage-specific manner, mostly in females, which exhibited a masculinized expression profile. This included 2 clock genes, Per2 and Arntl, implicating circadian circuits as being vulnerable to endocrine disruption. DNA methylation analysis of 2 genes, Per2 and Ar, showed no effect of EDCs and suggested alternative mechanisms for the altered mRNA levels. In the ARC, 12 genes were affected by treatment, mostly in males, again with dynamic developmental changes. Bionetwork analysis of relationships among genes, hormones, and physiological markers showed sexually dimorphic effects of estrogenic EDC exposures, with the female AVPV and the male ARC being most vulnerable, and provided novel relationships among hypothalamic genes and postnatal reproductive maturation.

Protein tyrosine kinase regulation by ubiquitination: critical roles of Cbl-family ubiquitin ligases.

Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell-cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant "activated PTK-selective" ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease.