The BTG2-PRMT1 module limits pre-B cell expansion by regulating the CDK4-Cyclin-D3 complex.

Developing pre-B cells in the bone marrow alternate between proliferation and differentiation phases. We found that protein arginine methyl transferase 1 (PRMT1) and B cell translocation gene 2 (BTG2) are critical components of the pre-B cell differentiation program. The BTG2-PRMT1 module induced a cell-cycle arrest of pre-B cells that was accompanied by re-expression of Rag1 and Rag2 and the onset of immunoglobulin light chain gene rearrangements. We found that PRMT1 methylated cyclin-dependent kinase 4 (CDK4), thereby preventing the formation of a CDK4-Cyclin-D3 complex and cell cycle progression. Moreover, BTG2 in concert with PRMT1 efficiently blocked the proliferation of BCR-ABL1-transformed pre-B cells in vitro and in vivo. Our results identify a key molecular mechanism by which the BTG2-PRMT1 module regulates pre-B cell differentiation and inhibits pre-B cell leukemogenesis.

ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.

The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

Targeted disruption of the BCR-ABL fusion gene by Cas9/dual-sgRNA inhibits proliferation and induces apoptosis in chronic myeloid leukemia cells.

The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein ( BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase ( ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.

A novel imatinib-upregulated long noncoding RNA plays a critical role in inhibition of tumor growth induced by Abl oncogenes.

BACKGROUND: Dysregulation of long noncoding RNAs (lncRNAs) has been linked to various human cancers. Bcr-Abl oncogene that results from a reciprocal translocation between human chromosome 9 and 22, is associated with several hematological malignancies. However, the role of lncRNAs in Bcr-Abl-induced leukemia remains largely unexplored. METHODS: LncRNA cDNA microarray was employed to identify key lncRNAs involved in Bcr-Abl-mediated cellular transformation. Abl-transformed cell survival and xenografted tumor growth in mice were evaluated to dissect the role of imatinib-upregulated lncRNA 1 (IUR1) in Abl-induced tumorigenesis. Primary bone marrow transformation and in vivo leukemia transplant using lncRNA-IUR1 knockout (KO) mice were further conducted to address the functional relevance of lncRNA-IUR1 in Abl-mediated leukemia. Transcriptome RNA-seq and Western blotting were performed to determine the mechanisms by which lncRNA-IUR1 regulates Bcr-Abl-induced tumorigenesis. RESULTS: We identified lncRNA-IUR1 as a critical negative regulator of Bcr-Abl-induced tumorigenesis. LncRNA-IUR1 expressed in a very low level in Bcr-Abl-positive cells from chronic myeloid leukemia patients. Interestingly, it was significantly induced in Abl-positive leukemic cells treated by imatinib. Depletion of lncRNA-IUR1 promoted survival of Abl-transformed human leukemic cells in experiments in vitro and xenografted tumor growth in mice, whereas ectopic expression of lncRNA-IUR1 sensitized the cells to apoptosis and suppressed tumor growth. In concert, silencing murine lncRNA-IUR1 in Abl-transformed cells accelerated cell survival and the development of leukemia in mice. Furthermore, lncRNA-IUR1 deficient mice were generated, and we observed that knockout of murine lncRNA-IUR1 facilitated Bcr-Abl-mediated primary bone marrow transformation. Moreover, animal leukemia model revealed that lncRNA-IUR1 deficiency promoted Abl-transformed cell survival and development of leukemia in mice. Mechanistically, we demonstrated that lncRNA-IUR1 suppressed Bcr-Abl-induced tumorigenesis through negatively regulating STAT5-mediated GATA3 expression. CONCLUSIONS: These findings unveil an inhibitory role of lncRNA-IUR1 in Abl-mediated cellular transformation, and provide new insights into molecular mechanisms underlying Abl-induced leukemogenesis.

Simultaneous Quantification of BCR-ABL and Bruton Tyrosine Kinase Inhibitors in Dried Plasma Spots and Its Application to Clinical Sample Analysis.

BACKGROUND: Recent reports highlight the importance of therapeutic drug monitoring (TDM) of BCR-ABL and Bruton tyrosine kinase inhibitors (TKIs); thus, large-scale studies are needed to determine the target concentrations of these drugs. TDM using dried plasma spots (DPS) instead of conventional plasma samples is a promising approach. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of BCR-ABL and Bruton TKIs for further TDM studies. METHODS: A 20-µL aliquot of plasma was spotted onto a filter paper and dried completely. Analytes were extracted from 2 DPS using 250 µL of solvent. After cleanup by supported liquid extraction, the sample was analyzed by LC-MS/MS. Applicability of the method was examined using samples of patients' DPS transported by regular mail as a proof-of-concept study. The constant bias and proportional error between plasma and DPS concentrations were assessed by Passing-Bablok regression analysis, and systematic errors were evaluated by Bland-Altman analysis. RESULTS: The method was successfully validated over the following calibration ranges: 1-200 ng/mL for dasatinib and ponatinib, 2-400 ng/mL for ibrutinib, 5-1000 ng/mL for bosutinib, and 20-4000 ng/mL for imatinib and nilotinib. TKI concentrations were successfully determined for 93 of 96 DPS from clinical samples. No constant bias between plasma and DPS concentrations was observed for bosutinib, dasatinib, nilotinib, and ponatinib, whereas there were proportional errors between the plasma and DPS concentrations of nilotinib and ponatinib. Bland-Altman plots revealed that significant systematic errors existed between both methods for bosutinib, nilotinib, and ponatinib. CONCLUSIONS: An LC-MS/MS method for the simultaneous quantification of 6 TKIs in DPS was developed and validated. Further large-scale studies should be conducted to assess the consistency of concentration measurements obtained from plasma and DPS.

c-Abl-Mediated Tyrosine Phosphorylation of PARP1 Is Crucial for Expression of Proinflammatory Genes.

Poly(ADP-ribosyl)ation is a rapid and transient posttranslational protein modification mostly catalyzed by poly(ADP-ribose) polymerase-1 (PARP1). Fundamental roles of activated PARP1 in DNA damage repair and cellular response pathways are well established; however, the precise mechanisms by which PARP1 is activated independent of DNA damage, and thereby playing a role in expression of inflammatory genes, remain poorly understood. In this study, we show that, in response to LPS or TNF-α exposure, the nonreceptor tyrosine kinase c-Abl undergoes nuclear translocation and interacts with and phosphorylates PARP1 at the conserved Y829 site. Tyrosine-phosphorylated PARP1 is required for protein poly(ADP-ribosyl)ation of RelA/p65 and NF-κB-dependent expression of proinflammatory genes in murine RAW 264.7 macrophages, human monocytic THP1 cells, or mouse lungs. Furthermore, LPS-induced airway lung inflammation was reduced by inhibition of c-Abl activity. The present study elucidated a novel signaling pathway to activate PARP1 and regulate gene expression, suggesting that blocking the interaction of c-Abl with PARP1 or pharmaceutical inhibition of c-Abl may improve the outcomes of PARP1 activation-mediated inflammatory diseases.

Interaction of the mTERT telomerase catalytic subunit with the c-Abl tyrosine kinase in mouse granulosa cells.

Context: Oocyte and granulosa cells (GCs) have bidirectional communication and GCs play an important role in folliculogenesis and proliferation of GCs is very important for the development of ovulatory follicle. DNA double-strand breaks activate c-Abl protein tyrosine kinase and c-Abl has a functional role in repairement of DNA and control of telomere.Objective: In this study, we hypothesized that c-Abl has a regulative role on mTERT in mouse ovarian granulosa cells (GCs) and we aimed to detect c-Abl and mTERT interaction in mouse primary culture of GCs.Materials and methods: Mouse ovarian granulosa cell were cultured and siRNA-mediated knockdown approach was used to knockdown c-Abl expression.Results: We showed c-Abl and mTERT immunolocalization in vivo and in vitro mouse GCs. c-Abl and mTERT were constitutively expressed in mouse granulosa cells and c-Abl presented more intense expression in granulosa cells than mTERT expression. The interaction of the c-Abl-mTERT is supported by the exhibition that c-Abl siRNA knockdown cells show decreased mTERT expression. We also present an interaction between c-Abl and mTERT by immunoprecipitation. In addition, our results indicated that the down-regulation of c-Abl was also accompanied by reduced expression of proliferating cell nuclear antigen (PCNA) in GCs.Conclusions: We suggest that mTERT may associate with the c-Abl in mouse GCs and the interactions between c-Abl and mTERT suggest a role for c-Abl in the regulation of telomerase function and proliferation in mouse granulosa cells.

T315I mutation exerts a dismal prognosis on adult BCR-ABL1-positive acute lymphoblastic leukemia, and salvage therapy with ponatinib or CAR-T cell and bridging to allogeneic hematopoietic stem cell transplantation can improve clinical outcomes.

A single-center retrospective was performed with consecutive de novo BCR-ABL1-positive acute lymphoblastic leukemia (ALL) patients who received TKI-containing therapy between January 2010 and December 2018 to review the incidence, treatment, and outcome of the T315I mutation. A total of 38 (18%) patients harbored the T315I mutation in this period. According to the type of salvage therapy, patients were divided into subgroups of hematopoietic stem cell transplantation (HSCT) recipients (n = 9) and HSCT nonrecipients (n = 29). In the latter subgroup, there were 7 patients who newly acquired the T315I mutation after HSCT, and the median time was 10.8 months. In addition to these 7 cases, 5 out of 22 patients were managed with chimeric antigen receptor (CAR) T cells and ponatinib. There were 4 patients in the HSCT recipient subgroup who were treated with CAR-T cells or ponatinib before HSCT. The complete molecular remission (CMR) and recurrence rate of HSCT recipients were both 67%, and the median recurrence time was 3.6 months. A better overall survival (OS) was observed in the HSCT recipient subgroup than in the HSCT nonrecipient subgroup (median of 12.3 months vs 3.3 months, respectively; p = 0.004). Compared with patients who were not bridging to HSCT, the patients who were treated with CAR-T cells and/or ponatinib and bridged to HSCT tended to have a better OS (median of 3.3 months vs 13.3, respectively; p = 0.09). In conclusion, the outcomes in ALL patients with the T315I BCR-ABL1 mutation were poor. A better OS can be achieved through ponatinib, CAR-T cells, and bridging to HSCT, but it also has a higher risk of recurrence.

Emodin Inhibits Resistance to Imatinib by Downregulation of Bcr-Abl and STAT5 and Allosteric Inhibition in Chronic Myeloid Leukemia Cells.

Imatinib-resistance is a significant concern for Bcr-Abl-positive chronic myelogenous leukemia (CML) treatment. Emodin, the predominant compound of traditional medicine rhubarb, was reported to inhibit the multidrug resistance by downregulating P-glycoprotein of K562/ADM cells with overexpression of P-glycoprotein in our previous studies. In the present study, we found that emodin can be a potential inhibitor for the imatinib-resistance in K562/G01 cells which are the imatinib-resistant subcellular line of human chronic myelogenous leukemia cells with overexpression of breakpoint cluster region-abelson (Bcr-Abl) oncoprotein. Emodin greatly enhanced cell sensitivity to imatinib, suppressed resistant cell proliferation and increased potentiated apoptosis induced by imatinib in K562/G01 cells. After treatment of emodin and imatinib together, the levels of p-Bcr-Abl and Bcr-Abl were significantly downregulated. Moreover, Bcr-Abl important downstream target, STAT5 and its phosphorylation were affected. Furthermore, the expression of Bcr-Abl and signal transducers and activators of transcription 5 (STAT5) related molecules, including c-MYC, MCL-1, poly(ADP-ribose)polymerase (PARP), Bcl-2 and caspase-3, were changed. Emodin also decreased Src expression and its phosphorylation. More importantly, emodin simultaneously targeted both the ATP-binding and allosteric sites on Bcr-Abl by molecular docking, with higher affinity with the myristoyl-binding site for enhanced Bcr-Abl kinase inhibition. Overall, these data indicated emodin might be an effective therapeutic agent for inhibiting resistance to imatinib in CML treatment.

T-cell blast crisis of chronic myelogenous leukemia presented with coexisting p210 and p190 BCR-ABL transcripts and t(10;11)(q11;p15).

BACKGROUND: Blast transformation of chronic myelogenous leukemia (CML) to T lymphoblastic lymphoma/acute lymphoblastic leukemia (T-LBL/ALL) is rare, and the molecular mechanism is still unclear. CASE REPORT: A 28-year-old woman who developed T-ALL with coexpressing both p210 and p190 BCR-ABL transcripts five years after the initial diagnosis of CML in chronic phase. The proliferation of bone marrow was extremely active with blast cells over 20%. Chromosome analysis revealed t(9;22)(q34;q11) and t(10;11)(q25;p15). Flow immunophenotyping showed that blasts expressed CD4, CD7, CD11b, CD38, CD34, CD33, and cCD3. CONCLUSION: It is the first T-cell blast of CML case with coexisting p210 and p190 as well as additional chromosome translocations. Through review this case and previous reports, we will reveal that CML patients with T-lymphocyte transformation depend on potential molecular and pathological mechanism.

An ultra-long circulating nanoparticle for reviving a highly selective BCR-ABL inhibitor in long-term effective and safe treatment of chronic myeloid leukemia.

Nanotechnology has demonstrated great promise for the development of more effective and safer cancer therapies. We recently developed a highly selective inhibitor of BCR-ABL fusion tyrosine kinase for chronic myeloid leukemia (CML). However, the poor drug-like properties were hurdles to its further clinical development. Herein, we re-investigate it by conjugating an amphiphilic polymer and self-assembling into a nanoparticle (NP) with a high loading (~10.3%). The formulation greatly improved its solubility and drastically extended its circulation half-life from ~5.3 to ~117 h (>20-fold). In the 150 days long-term engraftment model experiment, long intravenous dosing intervals of the NPs (every 4 or 8 days) exhibited much better survival and negligible toxicities as compared to daily oral administration of the inhibitor. Moreover, the NPs showed excellent inhibition of tumor growth in the subcutaneous xenograft model. All results suggest that the ultra-long circulating pro-drug NP may provide an effective and safe therapeutic strategy for BCR-ABL-positive CML.

Large amplicon droplet digital PCR for DNA-based monitoring of pediatric chronic myeloid leukaemia.

Quantification of tumour-specific molecular markers at the RNA and DNA level for treatment response monitoring is crucial for risk-adapted stratification and guidance of individualized therapy in leukaemia and other malignancies. Most pediatric leukaemias and solid tumours of mesenchymal origin are characterized by a relatively low mutation burden at the single nucleotide level and the presence of recurrent chromosomal translocations. The genomic fusion sites resulting from translocations are stable molecular tumour markers; however, repeat-rich DNA sequences flanking intronic breakpoints limit the design of high sensitivity PCR assays for minimal residual disease (MRD) monitoring. Here, we quantitatively evaluated the impact of repeat elements on assay selection and the feasibility of using extended amplicons (≤1330 bp) amplified by droplet digital PCR to monitor pediatric chronic myeloid leukaemia (CML). Molecular characterization of 178 genomic BCR-ABL1 fusion sites showed that 64% were located within sequence repeat elements, impeding optimal primer/probe design. Comparative quantification of DNA and RNA BCR-ABL1 copy numbers in 687 specimens from 55 pediatric patients revealed that their levels were highly correlated. The combination of droplet digital PCR, double quenched probes and extended amplicons represents a valuable tool for sensitive MRD assessment in CML and may be adapted to other translocation-positive tumours.

Philadelphia chromosome-positive acute lymphoblastic leukemia at first relapse in the era of tyrosine kinase inhibitors.

Despite the advances in the management of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) with the introduction of tyrosine kinase inhibitors (TKIs), relapses remain challenging. We reviewed clinical data from adult patients with Ph + ALL who received frontline hyperCVAD chemotherapy with a TKI to determine their outcomes after first relapse. Patients with first morphological relapse after prior complete remission were evaluated for predictors of response and survival. For 57 of 233 (25%) patients, there was morphological relapse after a median of 15.9 months from first remission [range: 5.3-94]. The choice of salvage treatments was at the discretion of the treating physician. So, 43 (75%) patients received a TKI in combination with their salvage treatment. Second remission was achieved in 41 of 49 (84%) evaluable patients. Median relapse free survival (RFS) was 10.5 months [range, 0.2-81]. The 1-year and 2-year overall survival (OS) were 41% and 20% respectively. On multivariate analysis, only elevated LDH (units/L), the use of first-generation or no TKI at the time of first relapse and the achievement of a major molecular response (MMR) had a significant effect on OS (HR: 2.82, 95% CI:1.11-7.16, P = .029; HR = 2.39, 95% CI: 1.07,5.39, P = .034; HR = 0.39, 95% CI: 0.16-0.94, P = .03, respectively). Whereas, only achievement of MMR was significantly prognostic for RFS with a HR of 0.48 (95% CI: 0.23-0.98, P = .04). The OS and RFS were comparable between recipients and non-recipients of allogeneic hematopoietic stem cell transplantation (alloHSCT) at second remission, due to a higher non-relapse mortality (53%) seen in patients who underwent alloHSCT.

Statistical algorithms improve accuracy of gene fusion detection.

Gene fusions are known to play critical roles in tumor pathogenesis. Yet, sensitive and specific algorithms to detect gene fusions in cancer do not currently exist. In this paper, we present a new statistical algorithm, MACHETE (Mismatched Alignment CHimEra Tracking Engine), which achieves highly sensitive and specific detection of gene fusions from RNA-Seq data, including the highest Positive Predictive Value (PPV) compared to the current state-of-the-art, as assessed in simulated data. We show that the best performing published algorithms either find large numbers of fusions in negative control data or suffer from low sensitivity detecting known driving fusions in gold standard settings, such as EWSR1-FLI1. As proof of principle that MACHETE discovers novel gene fusions with high accuracy in vivo, we mined public data to discover and subsequently PCR validate novel gene fusions missed by other algorithms in the ovarian cancer cell line OVCAR3. These results highlight the gains in accuracy achieved by introducing statistical models into fusion detection, and pave the way for unbiased discovery of potentially driving and druggable gene fusions in primary tumors.

Pre-B acute lymphoblastic leukaemia recurrent fusion, EP300-ZNF384, is associated with a distinct gene expression.

BACKGROUND: Zinc-finger protein 384 (ZNF384) fusions are an emerging subtype of precursor B-cell acute lymphoblastic leukaemia (pre-B-ALL) and here we further characterised their prevalence, survival outcomes and transcriptome. METHODS: Bone marrow mononuclear cells from 274 BCR-ABL1-negative pre-B-ALL patients were immunophenotyped and transcriptome molecularly characterised. Transcriptomic data was analysed by principal component analysis and gene-set enrichment analysis to identify gene and pathway expression changes. RESULTS: We exclusively detect E1A-associated protein p300 (EP300)-ZNF384 in 5.7% of BCR-ABL1-negative adolescent/young adult (AYA)/adult pre-B-ALL patients. EP300-ZNF384 patients do not appear to be a high-risk subgroup. Transcriptomic analysis revealed that EP300-ZNF384 samples have a distinct gene expression profile that results in the up-regulation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) and cell adhesion pathways and down-regulation of cell cycle and DNA repair pathways. CONCLUSIONS: Importantly, this report contributes to a better overview of the incidence of EP300-ZNF384 patients and show that they have a distinct gene signature with concurrent up-regulation of JAK-STAT pathway, reduced expression of B-cell regulators and reduced DNA repair capacity.

Direct Quantification of Trace Amounts of a Chronic Myeloid Leukemia Biomarker Using Locked Nucleic Acid Capture Probes.

Molecular monitoring is indispensable for the clinical management of chronic myeloid leukemia (CML) patients. Real-time quantitative polymerase chain reaction (RT-qPCR) is the gold standard for the quantitative assessment of BCR-ABL transcript levels, which are critical in clinical decision-making. However, the frequent recurrence of the disease after drug discontinuation for 60% of patients has necessitated more sensitive and specific techniques to detect residual BCR-ABL transcripts. Here, we describe a quantification method for the detection of BCR-ABL targets at very low concentrations (<10 copies/sample) in the presence of a million copies of normal BCR and ABL genes. In this method, a fully modified locked nucleic acid (LNA) and a LNA/DNA chimera were used as capture probes, and the quantitative imaging mode of atomic force microscopy (AFM) was employed. Targets with one of the major breakpoints (found in more than 95% of CML patients), b3a2 and b2a2, were quantified. The BCR-ABL target captured on a miniaturized LNA-probe spot was scanned at nanometric resolution, and the samples containing one to ten copies of the BCR-ABL genes were examined. It was observed that the highest sensitivity, i.e., the detection of a single copy of the target gene, could be achieved through multiple runs, and the observed cluster number was well correlative (adjusted R2 = 0.999) to the target copy number in the sample solution. This observation clearly demonstrates that the LNA-based platform is effective in quantifying BCR-ABL targets with extremely low copy numbers, highlighting the potential applicability of AFM for use in the direct quantification of such targets without amplification or labeling.

Normal ABL1 is a tumor suppressor and therapeutic target in human and mouse leukemias expressing oncogenic ABL1 kinases.

Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.

YAP promotes myogenic differentiation via the MEK5-ERK5 pathway.

Yes-associated protein (YAP) is a transcriptional coactivator in the Hippo pathway that regulates cell proliferation, differentiation, and apoptosis. The MEK5/ERK5 MAPK cascade is essential for the early step of myogenesis. In this study, we generated C2C12 stable cell lines that expressed YAP (C2C12-YAP cells) and found that ERK5 and MEK5 were activated in C2C12-YAP cells compared with control C2C12 (C2C12-vector) cells. C2C12-YAP stable cells also differentiated into myotubes better than C2C12-vector cells, and expressed elevated levels of myogenin, a transcription factor that regulates myogenesis, as well as elevated levels of myosin heavy chain, a skeletal muscle marker. Western blot analysis revealed that Src and c-Abl (Abelson murine leukemia viral oncogene homolog 1) activation were enhanced in C2C12-YAP cells. Conversely, treatment of inhibitors of c-Abl, Src, or MEK5 inhibited activation of MEK5 and ERK5 and myogenesis of C2C12 myoblasts. Specific interactions between YAP and proteins in the ERK5 pathway, such as MEK kinase 3 (MEKK3) and ERK5, were illustrated by coimmunoprecipitation experiments. MEKK3 contains the PPGY motif (aa 178-181), which may interact with YAP. Site-directed mutagenesis experiments revealed that expression of MEKK3 Y181F mutant inhibited MEK5/ERK5 activation and myogenic differentiation. These results suggest that YAP promotes muscle differentiation by activating the Abl/Src/MEKK3/MEK5/ERK5 kinase cascade.-Chen, T.-H., Chen, C.-Y., Wen, H.-C., Chang, C.-C., Wang, H.-D., Chuu, C.-P., Chang, C.-H. YAP promotes myogenic differentiation via the MEK5-ERK5 pathway.

Mean residual life regression with functional principal component analysis on longitudinal data for dynamic prediction.

Predicting patient life expectancy is of great importance for clinicians in making treatment decisions. This prediction needs to be conducted in a dynamic manner, based on longitudinal biomarkers repeatedly measured during the patient's post-treatment follow-up period. The prediction is updated any time a new biomarker measurement is obtained. The heterogeneity across patients of biomarker trajectories over time requires flexible and powerful approaches to model noisy and irregularly measured longitudinal data. In this article, we use functional principal component analysis (FPCA) to extract the dominant features of the biomarker trajectory of each individual, and use these features as time-dependent predictors (covariates) in a transformed mean residual life (MRL) regression model to conduct dynamic prediction. Simulation studies demonstrate the improved performance of the transformed MRL model that includes longitudinal biomarker information in the prediction. We apply the proposed method to predict the remaining time expectancy until disease progression for patients with chronic myeloid leukemia, using the transcript levels of an oncogene, BCR-ABL.

Breakpoint Cluster Region-Mediated Inflammation Is Dependent on Casein Kinase II.

The breakpoint cluster region (BCR) is known as a kinase and cause of leukemia upon fusing to Abl kinase. In this study, we demonstrate that BCR associated with the α subunit of casein kinase II (CK2α), rather than BCR itself, is required for inflammation development. We found that BCR knockdown inhibited NF-κB activation in vitro and in vivo. Computer simulation, however, suggested that the putative BCR kinase domain has an unstable structure with minimal enzymatic activity. Liquid chromatography-tandem mass spectrometry analysis showed that CK2α associated with BCR. We found the BCR functions are mediated by CK2α. Indeed, CK2α associated with adaptor molecules of TNF-αR and phosphorylated BCR at Y177 to establish a p65 binding site after TNF-α stimulation. Notably, p65 S529 phosphorylation by CK2α creates a p300 binding site and increased p65-mediated transcription followed by inflammation development in vivo. These results suggest that BCR-mediated inflammation is dependent on CK2α, and the BCR-CK2α complex could be a novel therapeutic target for various inflammatory diseases.