CALR-mutated cells are vulnerable to combined inhibition of the proteasome and the endoplasmic reticulum stress response.

Cancer is driven by somatic mutations that provide a fitness advantage. While targeted therapies often focus on the mutated gene or its direct downstream effectors, imbalances brought on by cell-state alterations may also confer unique vulnerabilities. In myeloproliferative neoplasms (MPN), somatic mutations in the calreticulin (CALR) gene are disease-initiating through aberrant binding of mutant CALR to the thrombopoietin receptor MPL and ligand-independent activation of JAK-STAT signaling. Despite these mechanistic insights into the pathogenesis of CALR-mutant MPN, there are currently no mutant CALR-selective therapies available. Here, we identified differential upregulation of unfolded proteins, the proteasome and the ER stress response in CALR-mutant hematopoietic stem cells (HSCs) and megakaryocyte progenitors. We further found that combined pharmacological inhibition of the proteasome and IRE1-XBP1 axis of the ER stress response preferentially targets Calr-mutated HSCs and megakaryocytic-lineage cells over wild-type cells in vivo, resulting in an amelioration of the MPN phenotype. In serial transplantation assays following combined proteasome/IRE1 inhibition for six weeks, we did not find preferential depletion of Calr-mutant long-term HSCs. Together, these findings leverage altered proteostasis in Calr-mutant MPN to identify combinatorial dependencies that may be targeted for therapeutic benefit and suggest that eradicating disease-propagating Calr-mutant LT-HSCs may require more sustained treatment.

Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation.

Somatic mutations in cancer genes have been detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, because mutated and wild-type cells are admixed, we have limited ability to link genotypes with phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing genotype, transcriptomes and methylomes in progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations result in myeloid over lymphoid bias, and an expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate, with dysregulated expression of lineage and leukemia stem cell markers. Mutated DNMT3A leads to preferential hypomethylation of polycomb repressive complex 2 targets and a specific CpG flanking motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics paves the road to defining the downstream consequences of mutations that drive clonal mosaicism.

GATA1 downregulation in prefibrotic and fibrotic stages of primary myelofibrosis and in the myelofibrotic progression of other myeloproliferative neoplasms.

GATA binding protein 1 (GATA1) is a transcription factor essential for effective erythropoiesis and megakaryopoiesis. Two isoforms of GATA1 exist, derived from alternative splicing. "GATA1" is the full length and functionally active protein; "GATA1s" is the truncated isoform devoid of the activation domain, the function of which has not been fully elucidated. Reduced megakaryocytic expression of GATA1 has been linked to impaired hematopoiesis and bone marrow fibrosis in murine models and in vivo in patients affected by primary myelofibrosis (PMF). However, data is limited regarding GATA1 expression in other myeloproliferative neoplasms (MPN) such as pre-fibrotic PMF (pre-PMF), polycythemia vera (PV) and essential thrombocythemia (ET) and in their respective fibrotic progression. To assess whether an immunohistologic approach can be of help in separating different MPN, we have performed a comprehensive immunohistochemical evaluation of GATA1 expression in megakaryocytes within a cohort of BCR-ABL1 negative MPN. In order to highlight any potential differences between the two isoforms we tested two clones, one staining the sum of GATA1 and GATA1s ("clone 1"), the other staining GATA1 full length alone ("clone 2"). At the chronic phase, a significant reduction preferentially of GATA1 full length was seen in pre-fibrotic PMF, particularly compared to ET and PV; no significant differences were observed between PV and ET. The fibrotic progression of both PV and ET was associated with a significant reduction in GATA1, particularly affecting the GATA1 full length isoform. The fibrotic progression of pre-PMF to PMF was associated with a significant reduction of the overall GATA1 protein and a trend in reduction of GATA1s. Our findings support a role of GATA1 in the pathogenesis of BCR-ABL1 negative MPN, particularly in their fibrotic progression and suggest that the immunohistochemical evaluation of GATA1 may be of use in the differential diagnosis of these neoplasms.

Integrating genetic and non-genetic determinants of cancer evolution by single-cell multi-omics.

Cancer represents an evolutionary process through which growing malignant populations genetically diversify, leading to tumour progression, relapse and resistance to therapy. In addition to genetic diversity, the cell-to-cell variation that fuels evolutionary selection also manifests in cellular states, epigenetic profiles, spatial distributions and interactions with the microenvironment. Therefore, the study of cancer requires the integration of multiple heritable dimensions at the resolution of the single cell - the atomic unit of somatic evolution. In this Review, we discuss emerging analytic and experimental technologies for single-cell multi-omics that enable the capture and integration of multiple data modalities to inform the study of cancer evolution. These data show that cancer results from a complex interplay between genetic and non-genetic determinants of somatic evolution.

DNA methylation disruption reshapes the hematopoietic differentiation landscape.

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies1-3 and clonal hematopoiesis4,5. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid versus myelomonocytic progenitors following Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells. To reconcile genome-wide DNAme changes with specific erythroid versus myelomonocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the topography of hematopoietic differentiation, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in CpG enrichment of the transcription factor binding motif.

Somatic mutations and cell identity linked by Genotyping of Transcriptomes.

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.

TARGET-seq Takes Aim at Cancer Evolution through Multi-omics Single-Cell Genotyping and Transcriptomics.

In this issue of Molecular Cell, Rodriguez-Meira et al. (2019) present TARGET-seq, an elegant single-cell method that genotypes somatic mutations and captures whole transcriptomes in the same tumor cells, thus paving the way to directly link somatic mutations with resulting transcriptional phenotypes in clonally diverse cancer populations.

Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum.

Some secreted proteins that assemble into large complexes, such as extracellular matrices or hormones and enzymes in storage granules, must be kept at subaggregation concentrations during intracellular trafficking. We show surfeit locus protein 4 (Surf4) is the cargo receptor that establishes different steady-state concentrations for a variety of soluble cargo proteins within the endoplasmic reticulum (ER) through interaction with the amino-terminal tripeptides exposed after removal of leader sequences. We call this motif the ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif (ER-ESCAPE motif). Proteins that most readily aggregate in the ER lumen (e.g., dentin sialophosphoprotein [DSPP] and amelogenin, X-linked [AMELX]) have strong ER-ESCAPE motifs to inhibit aggregate formation, while less susceptible cargo exhibits weaker motifs. Specific changes in a single amino acid of the tripeptide result in aggregate formation and failure to efficiently traffic cargo out of the ER. A logical subset of 8,000 possible tripeptides starting a model soluble cargo protein (growth hormone) established a continuum of steady-state ER concentrations ranging from low (i.e., high affinity for receptor) to the highest concentrations associated with bulk flow-limited trafficking observed for nonbinding motifs. Human cells lacking Surf4 no longer preferentially trafficked cargo expressing strong ER-ESCAPE motifs. Reexpression of Surf4 or expression of yeast's ortholog, ER-derived vesicles protein 29 (Erv29p), rescued enhanced ER trafficking in Surf4-null cells. Hence our work describes a new way of preferentially exporting soluble cargo out of the ER that maintains proteins below the concentrations at which they form damaging aggregates.

Assessment of the Utility of Cytology and Flow Cytometry of Cerebrospinal Fluid Samples in Clinical Practice.

OBJECTIVES: We sought to assess the utility and limitations of both flow cytometry (FC) and cytology for the analysis of cerebrospinal fluid (CSF) in a practical clinical setting. METHODS: A total of 393 consecutive CSF samples from 171 patients submitted for both cytomorphologic and FC assessments were analyzed. RESULTS: Both FC and cytology findings were negative for malignancy in 315/393 samples (80%), and either positive (POS) or suspicious/atypical (SUSP/AT) in 7% of samples. This resulted in high agreement between FC and cytology (87%). Minor discrepancies were present in 4% of the cases. In 28 samples, an abnormal population was detected by FC but not by cytology. CONCLUSIONS: FC and cytology are important complementary methods for analyzing CSF samples. In cases where cytology is SUSP/AT and FC is inconclusive or negative, additional specimens should be submitted for immunostaining, cytogenetics, and/or molecular studies.

Cytologic diagnosis of adrenal oncocytic pheochromocytoma in a lung cancer patient: Report of a case and review of the literature.

Adrenal oncocytic pheochromocytoma is an extremely rare type of pheochromocytoma. To the best of our knowledge, we present the first cytological diagnosis of this variant via fine-needle aspiration in an 81-year-old male patient who was found to have an adrenal mass while undergoing workup of the recently diagnosed lung adenocarcinoma. We describe the cytomorphologic findings in our case and provide a review of the reported cases of adrenal oncocytic pheochromocytoma - all of which appear to be benign, nonfunctional, occur in adults, and have similar morphologic features. The pathologist should be aware of this uncommon diagnostic entity and its potential diagnostic pitfalls.

Efficient trafficking of acidic proteins out of the endoplasmic reticulum involves a conserved amino terminal IleProVal (IPV)-like tripeptide motif.

Most of the proposed extracellular biomineralization processes include the secretion of proteins that interact with mineral ions and/or mineral surfaces. Typically these proteins are acidic or have acidic domains that interact with multivalent cations in the extracellular environment. We propose that most acidic, Ca(2+)-binding proteins challenge the cell's mechanisms for trafficking through the endoplasmic reticulum (ER) lumen due to lumenal mM calcium that cause them to form large aggregates. We have recently shown that >95% of the DSPP mutations that cause non-syndromic genetic dentin diseases start their dominant negative affects by failing to rapidly exit the ER likely by forming complexes that cannot be normally trafficked to the Golgi. The complexes of mutant DSPP then capture more (severe disease) or less (mild disease) of the DSPP translated from the normal allele. After searching genomic databases as well as the published literature, we found the IleProVal (IPV)-like motif at the predicted amino terminus of many acidic proteins made in the mineralizing as well as non-mineralizing tissues of many species including vertebrates, echinoderms, mollusks, and yeast. While we often focused on acidic proteins reported associated with mineralizing structures, proteins associated with hormones and their storage/secretion, digestion, blood functions, as well as milk and other secreted fluids started with variations of the motif. Our hypothesis is that the IPV-like motif interacts with a highly conserved cargo receptor in the ER that efficiently traffics the acidic proteins out of the organelle before they can form harmful aggregates in the Ca(2+)-rich lumen.

Interleukin-6 expands homeostatic space for peripheral T cells.

T cell homeostasis and survival is dependent on interleukin-7 (IL-7). Immune activation, however, downregulates IL-7 receptor expression on T cells so that T cell survival during activation must be maintained independently of IL-7. The pro-inflammatory cytokine IL-6 shares common signaling pathways with IL-7 and can promote T cell survival in vitro. But whether IL-6 promotes T cell survival and homeostasis in vivo is not clear. Notably, IL-6 overexpression results in massive plasmacytosis and autoimmunity so that an IL-6 effect on in vivo T cell survival has remained untested. To overcome this limitation, here we generated IL-6 transgenic mice on an immunoglobulin heavy chain (IgH) deficient background which rendered them B cell deficient. Notably, such IgH(KO)IL6(Tg) mice were free of any signs of inflammation or autoimmunity and remained healthy throughout the course of analysis. In these mice, we found that IL-6 overexpression significantly increased peripheral T cell numbers, but importantly without increasing thymopoiesis. Moreover, IL-6 signaled T cells maintained their naïve phenotype and did not express activation/memory markers, suggesting that increased T cell numbers were due to increased T cell survival and not because of expansion of activated T cells. Mechanistically, we found that IL-6 signaling induced expression of pro-survival factors Mcl-1 and Pim-1/-2 but not Bcl-2. Thus, IL-6 is a T cell homeostatic cytokine that expands T cell space and can maintain the naïve T cell pool.

Pim1 permits generation and survival of CD4+ T cells in the absence of γc cytokine receptor signaling.

γ-Chain (γc) cytokine receptor signaling is required for the development of all lymphocytes. Why γc signaling plays such an essential role is not fully understood, but induction of the serine/threonine kinase Pim1 is considered a major downstream event of γc as Pim1 prevents apoptosis and increases metabolic activity. Consequently, we asked whether Pim1 overexpression would suffice to restore lymphocyte development in γc-deficient mice. By analyzing Pim1-transgenic γc-deficient mice (Pim1(Tg) γc(KO) ), we show that Pim1 promoted T-cell development and survival in the absence of γc. Interestingly, such effects were largely limited to CD4(+) lineage αß T cells as CD4(+) T-cell numbers improved to near normal levels but CD8(+) T cells remained severely lymphopenic. Notably, Pim1 over-expression failed to promote development and survival of any T-lineage cells other than αß T cells, as we observed complete lack of γδ, NKT, FoxP3(+) T regulatory cells and TCR-ß(+) CD8αα IELs in Pim1(Tg) γc(KO) mice. Collectively, these results uncover distinct requirements for γc signaling between CD4(+) αß T cells and all other T-lineage cells, and they identify Pim1 as a novel effector molecule sufficient to drive CD4(+) αß T-cell development and survival in the absence of γc cytokine receptor signaling.

Expression and possible role of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in the human endometrium and endometriosis.

BACKGROUND: Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) is involved in cellular processes such as inflammation, apoptosis and tumorigenesis. However, little is known about the expression and function of NAG-1 in the endometrium. This study aimed to evaluate the expression of NAG-1 in the endometrium and in the absence or presence of endometriosis and to investigate the effect of celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, on NAG-1 mRNA levels and apoptosis in human endometrial stromal cells (HESCs). METHODS: Eutopic endometrial samples were obtained during surgery from 40 patients with, and 40 patients without, endometriosis. Real-time PCR was used to quantify NAG-1 mRNA levels and immunohistochemistry was used to localize NAG-1 protein in the endometrium. To investigate the effects of celecoxib, HESCs were isolated and cultured with different concentrations of celecoxib or with 100 µM celecoxib at different times. Apoptosis was assessed by flow cytometry. RESULTS: NAG-1 mRNA levels and immunoreactivity showed cyclical changes through the menstrual cycle, increasing during the late secretory and menstrual phases. NAG-1 mRNA and protein levels were significantly lower in patients with endometriosis, compared with the control group. Celecoxib induced NAG-1 mRNA levels and apoptosis in cultured HESCs, with the effects dependent on drug concentrations and duration of treatment. Celecoxib treatment had no effect on prostaglandin E(2) levels in the culture supernatants. CONCLUSIONS: NAG-1 may be important in maintaining homeostasis in the normal endometrium and alterations in NAG-1 expression may be associated with the establishment of endometriosis. NAG-1 might be a therapeutic target for endometriosis.

Endometrial osteopontin mRNA expression and plasma osteopontin levels are increased in patients with endometriosis.

PROBLEM: The aim of this study was to evaluate osteopontin (OPN) mRNA expression in eutopic endometrium and plasma OPN levels in patients with endometriosis. METHOD OF STUDY: A total of 79 patients with histologically confirmed endometriosis and 43 patients without endometriosis participated in this study. OPN mRNA expression in endometrial tissues was measured by real-time quantitative polymerase chain reaction (PCR) and plasma concentrations of OPN were quantified using a specific commercial sandwich enzyme-linked immunosorbent assays (ELISA). RESULTS: Osteopontin mRNA expression in endometrial tissue was significantly higher in women with endometriosis than in controls (P = 0.010). The mean plasma levels of OPN (mean +/- S.E.M.) in patients with endometriosis and controls were 407.31 +/- 37.80 ng/mL and 165.84 +/- 19.29 ng/mL, respectively (P < 0.001). Receiver operating characteristic (ROC) analysis for plasma OPN revealed an area under the curve (AUC) of 0.894, with a sensitivity of 93.0%, specificity of 72.4%, positive likelihood ratio of 3.37, and negative likelihood ratio of 0.1 using a cut-off value of 167.68 ng/mL. CONCLUSION: Osteopontin may be involved in the pathogenesis of endometriosis and plasma OPN may be a useful non-invasive marker for the diagnosis of endometriosis.

Neutrophil-to-lymphocyte ratio as an adjunct to CA-125 for the diagnosis of endometriosis.

OBJECTIVE: To investigate the clinical value of differential white blood cell counts and neutrophil-to-lymphocyte ratio (NLR), by themselves or as adjunct to CA-125, in the diagnosis of endometriosis. DESIGN: Retrospective study. SETTING: University Medical Center. PATIENT(S): Two hundred thirty-one patients with endometriosis, 145 patients with benign ovarian tumors, and 384 healthy controls participated in this study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sensitivities and specificities of differential white blood cell (WBC) counts, NLR, serum CA-125, and the combined marker (NLR and serum CA-125) were evaluated by receiver-operating characteristic (ROC) analysis. RESULT(S): The mean NLR and the combined marker in patients with endometriosis were significantly higher than those in patients without endometriosis. The NLR was able to discriminate patients with endometriosis from those with benign ovarian tumors and from healthy controls, and 25 of the 38 endometriosis patients (65.8%) with minimal-to-mild disease exceeded the cutoff value. The combined marker had a sensitivity of 69.3% and specificity of 83.9% with a cutoff value of 55.7, showing 13.5% increase in sensitivity but 8.9% decrease in specificity when compared with serum CA-125 at a cutoff level of 35 IU/mL. The elevated combined marker detected 16 patients with endometriosis (42.1%) with minimal-to-mild disease, whereas only 10 patients (26.3%) had their serum CA-125 levels at more than 35 IU/mL. CONCLUSION(S): Measurement of NLR and the combined marker may be used as simple and easily obtained diagnostic markers for endometriosis.

Redox transformations of bis(2,2'-bipyridine)(1-methyl-1-pyridin-2-yl-ethylamine)ruthenium(II).

The amineruthenium(II) complex Ru(bpy)2(mpea)2+ has been prepared by the direct reaction of 1-methyl-1-pyridin-2-yl-ethylamine (mpea) with Ru(bpy)2Cl2 in ethanol/water and isolated as the hexafluorophosphate salt. Electrochemical analysis of this complex shows that it undergoes sequential one-electron oxidations to an amidoruthenium(III) intermediate (E degrees' = 1.086 V vs NHE) and then to an amidoruthenium(IV) (E degrees' = 0.928 V) or imidoruthenium(IV) (E degrees' = 1.083 V) complex, depending upon the solution pH (pKa = 2.62 for the amidoruthenium(IV) species). At higher potentials ( Epa = 1.5 V in 1.0 M H2SO4), the amido- or imidoruthenium(IV) species is irreversibly oxidized to the corresponding nitrosoruthenium(II) complex. The mechanism for this transformation appears, on the basis of b3lyp/cpcm/cep-31g(d) computations, to proceed through an imidoruthenium(V) intermediate, which is rapidly attacked by water to yield a Ru(II)-bound hydroxylamine radical, which is readily oxidized and deprotonated to produce the nitrosoruthenium(II) complex. The nitrosoruthenium(II) complex is quantitatively reduced to the original [Ru(bpy)2(mpea)]2+ complex at relatively negative potentials ( Epc = -0.2 V in 1.0 M H2SO4).