Predictive model for a second hip fracture occurrence using natural language processing and machine learning on electronic health records.

Hip fractures (HFx) are associated with a higher morbidity and mortality rates, leading to a significant reduction in life quality and in limitation of patient´s mobility. The present study aimed to obtain real-world evidence on the clinical characteristics of patients with an initial and a second hip fracture (HFx) and develop a predictive model for second HFx using artificial intelligence. Electronic health records from one hospital centre in Spain from January 2011 to December 2019 were analysed using EHRead® technology, based on natural language processing and machine learning. A total of 1,960 patients with HFx were finally included during the study period after meeting all inclusion and exclusion criteria. From this total, 1835 (93.6%) patients were included in the HFx subgroup, while 124 (6.4%) were admitted to the second HFx (2HFx) subgroup. The mean age of the participants was 84 years and 75.5% were female. Most of comorbidities were more frequently identified in the HFx group, including hypertension (72.0% vs. 67.2%), cognitive impairment (33.0% vs. 31.2%), diabetes mellitus (28.7% vs. 24.8%), heart failure (27.6% vs. 22.4%) and chronic kidney disease (26.9% vs. 16.0%). Based on clinical criteria, 26 features were selected as potential prediction factors. From there, 16 demographics and clinical characteristics such as comorbidities, medications, measures of disabilities for ambulation and type of refracture were selected for development of a competitive risk model. Specifically, those predictors with different associated risk ratios, sorted from higher to lower risk relevance were visual deficit, malnutrition, walking assistance, hypothyroidism, female sex, osteoporosis treatment, pertrochanteric fracture, dementia, age at index, osteoporosis, renal failure, stroke, COPD, heart disease, anaemia, and asthma. This model showed good performance (dependent AUC: 0.69; apparent performance: 0.75) and could help the identification of patients with higher risk of developing a second HFx, allowing preventive measures. This study expands the current available information of HFx patients in Spain and identifies factors that exhibit potential in predicting a second HFx among older patients.

Sex-Biased ZRSR2 Mutations in Myeloid Malignancies Impair Plasmacytoid Dendritic Cell Activation and Apoptosis.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDC). BPDCN occurs at least three times more frequently in men than in women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN, and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation. SIGNIFICANCE: Sex bias in cancer is well recognized, but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.This article is highlighted in the In This Issue feature, p. 275.

Small molecule inhibition of deubiquitinating enzyme JOSD1 as a novel targeted therapy for leukemias with mutant JAK2.

Mutations in the Janus Kinase 2 (JAK2) gene resulting in constitutive kinase activation represent the most common genetic event in myeloproliferative neoplasms (MPN), a group of diseases involving overproduction of one or more kinds of blood cells, including red cells, white cells, and platelets. JAK2 kinase inhibitors, such as ruxolitinib, provide clinical benefit, but inhibition of wild-type (wt) JAK2 limits their clinical utility due to toxicity to normal cells, and small molecule inhibition of mutated JAK2 kinase activity can lead to drug resistance. Here, we present a strategy to target mutated JAK2 for degradation, using the cell's intracellular degradation machinery, while sparing non-mutated JAK2. We employed a chemical genetics screen, followed by extensive selectivity profiling and genetic studies, to identify the deubiquitinase (DUB), JOSD1, as a novel regulator of mutant JAK2. JOSD1 interacts with and stabilizes JAK2-V617F, and inactivation of the DUB leads to JAK2-V617F protein degradation by increasing its ubiquitination levels, thereby shortening its protein half-life. Moreover, targeting of JOSD1 leads to the death of JAK2-V617F-positive primary acute myeloid leukemia (AML) cells. These studies provide a novel therapeutic approach to achieving selective targeting of mutated JAK2 signaling in MPN.

Chromatin accessibility promotes hematopoietic and leukemia stem cell activity.

Chromatin organization is a highly orchestrated process that influences gene expression, in part by modulating access of regulatory factors to DNA and nucleosomes. Here, we report that the chromatin accessibility regulator HMGN1, a target of recurrent DNA copy gains in leukemia, controls myeloid differentiation. HMGN1 amplification is associated with increased accessibility, expression, and histone H3K27 acetylation of loci important for hematopoietic stem cells (HSCs) and leukemia, such as HoxA cluster genes. In vivo, HMGN1 overexpression is linked to decreased quiescence and increased HSC activity in bone marrow transplantation. HMGN1 overexpression also cooperates with the AML-ETO9a fusion oncoprotein to impair myeloid differentiation and enhance leukemia stem cell (LSC) activity. Inhibition of histone acetyltransferases CBP/p300 relieves the HMGN1-associated differentiation block. These data nominate factors that modulate chromatin accessibility as regulators of HSCs and LSCs, and suggest that targeting HMGN1 or its downstream effects on histone acetylation could be therapeutically active in AML.

Trisomy of a Down Syndrome Critical Region Globally Amplifies Transcription via HMGN1 Overexpression.

Down syndrome (DS, trisomy 21) is associated with developmental abnormalities and increased leukemia risk. To reconcile chromatin alterations with transcriptome changes, we performed paired exogenous spike-in normalized RNA and chromatin immunoprecipitation sequencing in DS models. Absolute normalization unmasks global amplification of gene expression associated with trisomy 21. Overexpression of the nucleosome binding protein HMGN1 (encoded on chr21q22) recapitulates transcriptional changes seen with triplication of a Down syndrome critical region on distal chromosome 21, and HMGN1 is necessary for B cell phenotypes in DS models. Absolute exogenous-normalized chromatin immunoprecipitation sequencing (ChIP-Rx) also reveals a global increase in histone H3K27 acetylation caused by HMGN1. Transcriptional amplification downstream of HMGN1 is enriched for stage-specific programs of B cells and B cell acute lymphoblastic leukemia, dependent on the developmental cellular context. These data offer a mechanistic explanation for DS transcriptional patterns and suggest that further study of HMGN1 and RNA amplification in diverse DS phenotypes is warranted.

TNF Signaling through RIP1 Kinase Enhances SN38-Induced Death in Colon Adenocarcinoma.

Elucidation of TNF-directed mechanisms for cell death induction and maintenance of tumor growth has revealed a role for receptor-interacting protein kinases 1 and 3 (RIPK1/RIP1 and RIPK3/RIP3), components of the necrosome complex, as determinants of cell fate. Here, the participation of TNF signaling was analyzed with regard to the cytotoxic action of different DNA-damaging agents in a panel of colon cancer cells. While most of these cell lines were insensitive to TNF, combination with these drugs increased sensitivity by inducing cell death and DNA damage, especially in the case of the topoisomerase inhibitor SN38. Changes in levels of RIP1 and RIP3 occurred following monotherapy with SN38 or in combination with TNF. Downregulation of RIP1 resulted in increased resistance to SN38, implying a requirement for RIP1 in mediating cytotoxicity through the TNF/TNFR signaling pathway. Downregulation of RIP1 in a xenograft model impaired tumor growth inhibition from SN38 treatment, suggesting the potential of RIP1 to determine the clinical outcome of irinotecan treatment. These results indicate that TNF plays a key role in determining the cytotoxic effectiveness of SN38 in colorectal cancer and suggests a re-evaluation of TNF-based interventions to enhance therapeutic efficacy.Implications: The capacity of RIP1 to influence drug sensitivity suggests RIP1 may have biomarker potential. Mol Cancer Res; 15(4); 395-404. ©2017 AACR.

Blastic Plasmacytoid Dendritic Cell Neoplasm Is Dependent on BCL2 and Sensitive to Venetoclax.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy with dismal outcomes for which no standard therapy exists. We found that primary BPDCN cells were dependent on the antiapoptotic protein BCL2 and were uniformly sensitive to the BCL2 inhibitor venetoclax, as measured by direct cytotoxicity, apoptosis assays, and dynamic BH3 profiling. Animals bearing BPDCN patient-derived xenografts had disease responses and improved survival after venetoclax treatment in vivo Finally, we report on 2 patients with relapsed/refractory BPDCN who received venetoclax off-label and experienced significant disease responses. We propose that venetoclax or other BCL2 inhibitors undergo expedited clinical evaluation in BPDCN, alone or in combination with other therapies. In addition, these data illustrate an example of precision medicine to predict treatment response using ex vivo functional assessment of primary tumor tissue, without requiring a genetic biomarker. SIGNIFICANCE: Therapy for BPDCN is inadequate, and survival in patients with the disease is poor. We used primary tumor cell functional profiling to predict BCL2 antagonist sensitivity as a common feature of BPDCN, and demonstrated in vivo clinical activity of venetoclax in patient-derived xenografts and in 2 patients with relapsed chemotherapy-refractory disease. Cancer Discov; 7(2); 156-64. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115.

TRAF-mediated modulation of NF-kB AND JNK activation by TNFR2.

Tumor Necrosis Factor Receptor 2 (TNFR2) activates transcription factor κB (NF-κB) and c-Jun N-terminal kinase (JNK). Most of the biological activities triggered by TNFR2 depend on the recruitment of TNF Receptor-Associated Factor 2 (TRAF2) to the intracellular region of the receptor. The intracellular region of TNFR2 contains five highly conserved amino acid sequences, three of which appear implicated in receptor signaling. In this work we have studied the interaction of TNFR2 with TRAF proteins as well as the functional consequences of this interaction. We show that TRAF1, TRAF2 and TRAF3 bind to the receptor through two different binding sites located at conserved modules IV and V of its intracellular region, respectively. We also show that TRAF1 and TRAF3 have opposite effects to TRAF2 on NF-κB and JNK activation by TNFR2. Moreover, we show that TNFR2 is able to induce JNK activation in a TRAF2-independent fashion. This new receptor activity relies on a sequence located in the conserved module III. This region is also responsible for the ability of TNFR2 to induce TRAF2 degradation, thus emphasizing the role of conserved module III (amino acids 338-379) on receptor signaling and regulation. We show that only TNFR2 can induce TRAF2 degradation while TRAF1 or TRAF3 is not subjected to this regulatory mechanism and that TRAF1, but not TRAF3, is able to inhibit TRAF2 degradation.

A TRAF2 binding independent region of TNFR2 is responsible for TRAF2 depletion and enhancement of cytotoxicity driven by TNFR1.

Tumor Necrosis Factor (TNF) interacts with two receptors known as TNFR1 and TNFR2. TNFR1 activation may result in either cell proliferation or cell death. TNFR2 activates Nuclear Factor-kappaB (NF-kB) and c-Jun N-terminal kinase (JNK) which lead to transcriptional activation of genes related to cell proliferation and survival. This depends on the binding of TNF Receptor Associated Factor 2 (TRAF2) to the receptor. TNFR2 also induces TRAF2 degradation. In this work we have investigated the structural features of TNFR2 responsible for inducing TRAF2 degradation and have studied the biological consequences of this activity. We show that when TNFR1 and TNFR2 are co-expressed, TRAF2 depletion leads to an enhanced TNFR1 cytotoxicity which correlates with the inhibition of NF-kB. NF-kB activation and TRAF2 degradation depend of different regions of the receptor since TNFR2 mutants at amino acids 343-349 fail to induce TRAF2 degradation and have lost their ability to enhance TNFR1-mediated cell death but are still able to activate NF-kB. Moreover, whereas NF-kB activation requires TRAF2 binding to the receptor, TRAF2 degradation appears independent of TRAF2 binding. Thus, TNFR2 mutants unable to bind TRAF2 are still able to induce its degradation and to enhance TNFR1-mediated cytotoxicity. To test further this receptor crosstalk we have developed a system stably expressing in cells carrying only endogenous TNFR1 the chimeric receptor RANK-TNFR2, formed by the extracellular region of RANK (Receptor activator of NF-kB) and the intracellular region of TNFR2.This has made possible to study independently the signals triggered by TNFR1 and TNFR2. In these cells TNFR1 is selectively activated by soluble TNF (sTNF) while RANK-TNFR2 is selectively activated by RANKL. Treatment of these cells with sTNF and RANKL leads to an enhanced cytotoxicity.

Signal transduction by tumor necrosis factor receptors.

Tumor necrosis factor (TNF) is a key mediator in the inflammatory response which is implicated in the onset of a number of diseases. Research on TNF led to the characterization of the largest family of cytokines known until now, the TNF superfamily, which exert their biological effects through the interaction with transmembrane receptors of the TNFR superfamily. TNF itself exerts its biological effects interacting with two different receptors: TNFR1 and TNFR2. TNFR1 presents a death domain on its intracellular region. In contrast to TNFR1, TNFR2 does not have a death domain. Activation of TNFR1 implies the consecutive formation of two different TNF receptor signalling complexes. Complex I controls the expression of antiapoptotic proteins that prevent the triggering of cell death processes, whereas Complex II triggers cell death processes. TNFR2 only signals for antiapoptotic reactions. However, recent evidence indicates that TNFR2 also signals to induce TRAF2 degradation. TRAF2 is a key mediator in signal transduction of both TNFR1 and TNFR2. Thus, this novel signalling pathway has two important implications: on one hand, it represents an auto regulatory loop for TNFR2; on the other hand, when this signal is triggered TNFR1 activity is modified so that antiapoptotic pathways are inhibited and apoptotic reactions are enhanced.

NF-kappaB signal triggering and termination by tumor necrosis factor receptor 2.

Tumor necrosis factor receptor 2 (TNFR2) activates transcription factor κB (NF-κB) and c-Jun N-terminal kinase (JNK). The mechanisms mediating these activations are dependent on the recruitment of TNF receptor-associated factor 2 (TRAF2) to the intracellular region of the receptor. TNFR2 also induces TRAF2 degradation. We show that in addition to the well characterized TRAF2 binding motif 402-SKEE-405, the human receptor contains another sequence located at the C-terminal end (amino acids 425-439), which also recruits TRAF2 and activates NF-κB. In addition to that, human TNFR2 contains a conserved region (amino acids 338-379) which is responsible for TRAF2 degradation and therefore of terminating NF-κB signaling. TRAF2 degradation and the lack of NF-κB activation when both TNFR1 and TNFR2 are co-expressed results in an enhanced ability of TNFR1 to induce cell death, showing that the cross-talk between both receptors is of a great biological relevance. Induction of TRAF2 degradation appears to be independent of TRAF2 binding to the receptor. Amino acids 343-TGSSDSS-349 are essential for inducing TRAF2 degradation because deletion mutants of this region or point mutations at serine residues 345 and 346 or 348 and 349 obliterate the ability of TNFR2 to induce TRAF2 degradation.

Proteomic analysis of annexin A2 phosphorylation induced by microtubule interfering agents and kinesin spindle protein inhibitors.

Microtubule interfering agents (MIAs) are antitumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause the accumulation of mitotic cells and subsequently cell death. We used two-dimensional gel electrophoresis (2DE) followed by MALDI-MS analysis to demonstrate that the MIAs vinblastine (Velban) and paclitaxel (Taxol), as well as the KSP inhibitor S-tritil-L-cysteine, induce the phosphorylation of annexin A2 in human lung carcinoma A549 cells. Further tandem mass spectrometry analysis using a combination of peptide fragmentation methods (CID and ETD) and multiple reaction monitoring (MRM) analysis determined that this modification occurs mainly at threonine 19. We show that MIAs and KSP inhibitors only induce this phosphorylation in cells capable of reaching the M phase. Furthermore, we demonstrate that CDK activity is required for the phosphorylation of annexin A2 induced by MIAs and KSP inhibitors. Finally, we have used double thymidine block synchronization to demonstrate that annexin A2 is not phosphorylated during a normal mitosis, indicating that this phosphorylation of annexin A2 is a specific response to these drugs.

Microtubule interfering agents and KSP inhibitors induce the phosphorylation of the nuclear protein p54(nrb), an event linked to G2/M arrest.

Microtubule interfering agents (MIAs) are anti-tumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause G2/M arrest and cell death. Using 2D-PAGE followed by Nano-LC-ESI-Q-ToF analysis, we found that MIAs such as vincristine (Oncovin) or paclitaxel (Taxol) and KSP inhibitors such as S-tritil-l-cysteine induce the phosphorylation of the nuclear protein p54(nrb) in HeLa cells. Furthermore, we demonstrate that cisplatin (Platinol), an anti-tumor drug that does not cause M arrest, does not induce this modification. We show that the G2/M arrest induced by MIAs is required for p54(nrb) phosphorylation. Finally, we demonstrate that CDK activity is required for MIA-induced phosphorylation of p54(nrb).

Phosphorylation of human eukaryotic elongation factor 1Bgamma is regulated by paclitaxel.

Paclitaxel (Ptx) is an antitumoural drug that inhibits microtubule dynamics, causes G2/M arrest and induces cell death. 2-D PAGE and MALDI-TOF-MS analysis of HeLa cells extracts revealed that Ptx up-regulates a form of the eukaryotic elongation factor 1Bgamma (eEF1Bgamma) and down-regulates another one. This event, linked to the lack of Ptx effect over eEF1Bgamma mRNA or protein levels suggested a PTM of this elongation factor. Further 2-D PAGE analysis followed by a phosphospecific staining with PRO-Q Diamond showed the staining of the Ptx up-regulated form only. Moreover, this Ptx up-regulated form of eEF1Bgamma disappears upon treatment with protein phosphatase. Thus, we demonstrate that human eEF1Bgamma phosphorylation is regulated by Ptx.