Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A.

Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain-derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl-) cells through genome editing and compared HCV production between Abl- cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl-NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.

ABL allosteric inhibitors synergize with statins to enhance apoptosis of metastatic lung cancer cells.

Targeting mitochondrial metabolism has emerged as a treatment option for cancer patients. The ABL tyrosine kinases promote metastasis, and enhanced ABL signaling is associated with a poor prognosis in lung adenocarcinoma patients. Here we show that ABL kinase allosteric inhibitors impair mitochondrial integrity and decrease oxidative phosphorylation. To identify metabolic vulnerabilities that enhance this phenotype, we utilized a CRISPR/Cas9 loss-of-function screen and identified HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway and target of statin therapies, as a top-scoring sensitizer to ABL inhibition. Combination treatment with ABL allosteric inhibitors and statins decreases metastatic lung cancer cell survival in vitro in a synergistic manner. Notably, combination therapy in mouse models of lung cancer brain metastasis and therapy resistance impairs metastatic colonization with a concomitant increase in animal survival. Thus, metabolic combination therapy might be effective to decrease metastatic outgrowth, leading to increased survival for lung cancer patients with advanced disease.

Cortactin Is Required for Efficient FAK, Src and Abl Tyrosine Kinase Activation and Phosphorylation of Helicobacter pylori CagA.

Cortactin is a well-known regulatory protein of the host actin cytoskeleton and represents an attractive target of microbial pathogens like Helicobacter pylori. H. pylori manipulates cortactin's phosphorylation status by type-IV secretion-dependent injection of its virulence protein CagA. Multiple host tyrosine kinases, like FAK, Src, and Abl, are activated during infection, but the pathway(s) involved is (are) not yet fully established. Among them, Src and Abl target CagA and stimulate tyrosine phosphorylation of the latter at its EPIYA-motifs. To investigate the role of cortactin in more detail, we generated a CRISPR/Cas9 knockout of cortactin in AGS gastric epithelial cells. Surprisingly, we found that FAK, Src, and Abl kinase activities were dramatically downregulated associated with widely diminished CagA phosphorylation in cortactin knockout cells compared to the parental control. Together, we report here a yet unrecognized cortactin-dependent signaling pathway involving FAK, Src, and Abl activation, and controlling efficient phosphorylation of injected CagA during infection. Thus, the cortactin status could serve as a potential new biomarker of gastric cancer development.

Cumulative mechanism of several major imatinib-resistant mutations in Abl kinase.

Despite the outstanding success of the cancer drug imatinib, one obstacle in prolonged treatment is the emergence of resistance mutations within the kinase domain of its target, Abl. We noticed that many patient-resistance mutations occur in the dynamic hot spots recently identified to be responsible for imatinib's high selectivity toward Abl. In this study, we provide an experimental analysis of the mechanism underlying drug resistance for three major resistance mutations (G250E, Y253F, and F317L). Our data settle controversies, revealing unexpected resistance mechanisms. The mutations alter the energy landscape of Abl in complex ways: increased kinase activity, altered affinity, and cooperativity for the substrates, and, surprisingly, only a modestly decreased imatinib affinity. Only under cellular adenosine triphosphate (ATP) concentrations, these changes cumulate in an order of magnitude increase in imatinib's half-maximal inhibitory concentration (IC50). These results highlight the importance of characterizing energy landscapes of targets and its changes by drug binding and by resistance mutations developed by patients.

[Chronic myeloid leukemia harboring T315I and F317L mutations successfully treated with interferon-α and ponatinib].

Our patient was diagnosed with chronic myeloid leukemia (CML) in chronic phase (CP) when he was 40 years old. Although dasatinib (DAS) was prescribed during his clinical course, he was poorly compliant with the treatment. In November 20XX, at 65 years of age, he visited our hospital with leukocytosis. He was diagnosed with CML in CP and recommenced DAS at 50 mg/day, achieving a complete hematological response after 2 months. However, DAS was increased to 100 mg/day because only minimum cytogenetic response was evident even after 9 months, but CML progressed to the accelerated phase after 18 months. The ABL kinase domain mutations T315I and F317L were detected. Ponatinib (PON) was not yet approved, and he declined allogeneic stem cell transplantation therapy. He commenced interferon-α (IFN-α) in addition to DAS, and the F317L mutation (only) disappeared after 7 months; the patient achieved a major cytogenetic response. In January 20XX＋4, he commenced PON monotherapy (the drug was approved by this time) and achieved a major molecular response after 8 months. The T315I mutation disappeared during PON therapy. Although IFN-α is rarely used in the treatment of CML, this case suggests that IFN-α should be re-considered in patients with CML who exhibit tyrosine kinase inhibitor resistance.

Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma.

BACKGROUND: The mechanism underlying chromosome rearrangement in nasopharyngeal carcinoma (NPC) remains elusive. It is known that most of the aetiological factors of NPC trigger oxidative stress. Oxidative stress is a potent apoptotic inducer. During apoptosis, chromatin cleavage and DNA fragmentation occur. However, cells may undergo DNA repair and survive apoptosis. Non-homologous end joining (NHEJ) pathway has been known as the primary DNA repair system in human cells. The NHEJ process may repair DNA ends without any homology, although region of microhomology (a few nucleotides) is usually utilised by this DNA repair system. Cells that evade apoptosis via erroneous DNA repair may carry chromosomal aberration. Apoptotic nuclease was found to be associated with nuclear matrix during apoptosis. Matrix association region/scaffold attachment region (MAR/SAR) is the binding site of the chromosomal DNA loop structure to the nuclear matrix. When apoptotic nuclease is associated with nuclear matrix during apoptosis, it potentially cleaves at MAR/SAR. Cells that survive apoptosis via compromised DNA repair may carry chromosome rearrangement contributing to NPC tumourigenesis. The Abelson murine leukaemia (ABL) gene at 9q34 was targeted in this study as 9q34 is a common region of loss in NPC. This study aimed to identify the chromosome breakages and/or rearrangements in the ABL gene in cells undergoing oxidative stress-induced apoptosis. RESULTS: In the present study, in silico prediction of MAR/SAR was performed in the ABL gene. More than 80% of the predicted MAR/SAR sites are closely associated with previously reported patient breakpoint cluster regions (BCR). By using inverse polymerase chain reaction (IPCR), we demonstrated that hydrogen peroxide (H2O2)-induced apoptosis in normal nasopharyngeal epithelial and NPC cells led to chromosomal breakages within the ABL BCR that contains a MAR/SAR. Intriguingly, we detected two translocations in H2O2-treated cells. Region of microhomology was found at the translocation junctions. This observation is consistent with the operation of microhomology-mediated NHEJ. CONCLUSIONS: Our findings suggested that oxidative stress-induced apoptosis may participate in chromosome rearrangements of NPC. A revised model for oxidative stress-induced apoptosis mediating chromosome rearrangement in NPC is proposed.

c-Abl phosphorylation of Yin Yang 1's conserved tyrosine 254 in the spacer region modulates its transcriptional activity.

Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate for c-Abl kinase phosphorylation at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA, and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies reveal that c-Abl mediated phosphorylation of YY1 regulates YY1's transcriptional ability in vivo. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1's transcriptional activity, linking YY1 regulation with c-Abl tyrosine kinase signaling pathways.

Two novel fusion genes, AIF1L-ETV6 and ABL1-AIF1L, result together with ETV6-ABL1 from a single chromosomal rearrangement in acute lymphoblastic leukemia with prenatal origin.

Fusion genes resulting from chromosomal rearrangements represent a hallmark of childhood acute lymphoblastic leukemia (ALL). Unlike more common fusion genes generated via simple reciprocal chromosomal translocations, formation of the ETV6-ABL1 fusion gene requires 3 DNA breaks and usually results from an interchromosomal insertion. We report a child with ALL in which a single interchromosomal insertion led to the formation of ETV6-ABL1 and 2 novel fusion genes: AIF1L-ETV6 and ABL1-AIF1L. We demonstrate the prenatal origin of this complex chromosomal rearrangement, which apparently initiated the leukemogenic process, by successful backtracking of the ETV6-ABL1 fusion into the patient's archived neonatal blood. We cloned coding sequences of AIF1L-ETV6 and ABL1-AIF1L in-frame fusion transcripts from the patient's leukemic blasts and we show that the chimeric protein containing the DNA binding domain of ETV6 is expressed from the AIF1L-ETV6 transcript and localized in both the cytoplasm and nucleus of transfected HEK293T cells. Transcriptomic and genomic profiling of the diagnostic bone marrow sample revealed Ph-like gene expression signature and loss of the IKZF1 and CDKN2A/B genes, the typical genetic lesions accompanying ETV6-ABL1-positive ALL. The prenatal origin of the rearrangement confirms that ETV6-ABL1 is not sufficient to cause overt leukemia, even when combined with the 2 novel fusions. We did not find the AIF1L-ETV6 and ABL1-AIF1L fusions in other ETV6-ABL1-positive ALL. Nevertheless, functional studies would be needed to establish the biological role of AIF1L-ETV6 and ABL1-AIF1L and to determine whether they contribute to leukemogenesis and/or to the final leukemia phenotype.

Abl and Arg mediate cysteine cathepsin secretion to facilitate melanoma invasion and metastasis.

The incidence of melanoma is increasing, particularly in young women, and the disease remains incurable for many because of its aggressive, metastatic nature and its high rate of resistance to conventional, targeted, and immunological agents. Cathepsins are proteases that are critical for melanoma progression and therapeutic resistance. Intracellular cathepsins cleave or degrade proteins that restrict cancer progression, whereas extracellular cathepsins directly cleave the extracellular matrix and activate proinvasive proteases in the tumor microenvironment. Cathepsin secretion is markedly increased in cancer cells. We investigated the signaling pathways leading to increased cathepsin secretion in melanoma cells. We found that the nonreceptor tyrosine kinases Abl and Arg (Abl/Arg) promoted the secretion of cathepsin B and cathepsin L by activating transcription factors (namely, Ets1, Sp1, and NF-κB/p65) that have key roles in the epithelial-mesenchymal transition (EMT), invasion, and therapeutic resistance. In some melanoma cell lines, Abl/Arg promoted the Ets1/p65-induced secretion of cathepsin B and cathepsin L in a kinase-independent manner, whereas in other melanoma lines, Abl/Arg promoted the kinase-dependent, Sp1/Ets1/p65-mediated induction of cathepsin L secretion and the Sp1/p65-mediated induction of cathepsin B secretion. As an indication of clinical relevance, the abundance of mRNAs encoding Abl/Arg, Sp1, Ets1, and cathepsins was positively correlated in primary melanomas, and Abl/Arg-driven invasion in culture and metastasis in vivo required cathepsin secretion. These data suggest that drugs targeting Abl kinases, many of which are FDA-approved, might inhibit cathepsin secretion in some melanomas and potentially other aggressive cancers harboring activated Abl kinases.

ETV6-ABL1 fusion combined with monosomy 7 in childhood B-precursor acute lymphoblastic leukemia.

ETV6-ABL1 fusion is a rare but recurrent oncogenic lesion found in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), without an established chromosomal abnormality, and is associated with poor outcome. In ETV6-ABL1-positive cases, an in-frame fusion produced by a complex rearrangement results in constitutive chimeric tyrosine kinase activity. Monosomy 7 is also a rare and unfavorable chromosomal abnormality in childhood BCP-ALL. Here, we report a 14-year-old female BCP-ALL patient with ETV6-ABL1 fusion combined with monosomy 7. She was admitted to our hospital because of persistent fever. Bone marrow nuclear cell count on admission was 855,000/µL with 90.0% blastic cells of lymphoid morphology. Blasts were positive for CD10, CD19, CD20, CD34, cyCD79a, cyTdT, HLA-DR, and CD66c, had a karyotype of 45, XX, - 7 [18/20] and a split signal for ABL1 FISH probe (92.7%), and were sensitive to tyrosine kinase inhibitors, imatinib and dasatinib, in vitro. ETV6-ABL1 fusion transcript was identified by whole transcriptome sequencing and confirmed by RT-PCR. She was treated with the high-risk protocol based on ALL-BFM 95, achieved complete remission (CR) after induction chemotherapy, and maintained CR for 4 months. To our knowledge, this is the first report of ETV6-ABL1 fusion combined with monosomy 7 in childhood BCP-ALL.

G-Protein Gα13 Functions with Abl Kinase to Regulate Actin Cytoskeletal Reorganization.

Heterotrimeric G-proteins are essential cellular signal transducers. One of the G-proteins, Gα13, is critical for actin cytoskeletal reorganization, cell migration, cell proliferation, and apoptosis. Previously, we have shown that Gα13 is essential for both G-protein-coupled receptor and receptor tyrosine kinase-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. However, the mechanism by which Gα13 signals to actin cytoskeletal reorganization is not completely understood. Here we show that Gα13 directly interacts with Abl tyrosine kinase, which is a critical regulator of actin cytoskeleton. This interaction is critical for Gα13-induced dorsal ruffle turnover, endothelial cell remodeling, and cell migration. Our data uncover a new molecular signaling pathway by which Gα13 controls actin cytoskeletal reorganization.

Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination.

Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair.

Different BCR/Abl protein suppression patterns as a converging trait of chronic myeloid leukemia cell adaptation to energy restriction.

BCR/Abl protein drives the onset and progression of Chronic Myeloid Leukemia (CML). We previously showed that BCR/Abl protein is suppressed in low oxygen, where viable cells retain stem cell potential. This study addressed the regulation of BCR/Abl protein expression under oxygen or glucose shortage, characteristic of the in vivo environment where cells resistant to tyrosine kinase inhibitors (TKi) persist. We investigated, at transcriptional, translational and post-translational level, the mechanisms involved in BCR/Abl suppression in K562 and KCL22 CML cells. BCR/abl mRNA steady-state analysis and ChIP-qPCR on BCR promoter revealed that BCR/abl transcriptional activity is reduced in K562 cells under oxygen shortage. The SUnSET assay showed an overall reduction of protein synthesis under oxygen/glucose shortage in both cell lines. However, only low oxygen decreased polysome-associated BCR/abl mRNA significantly in KCL22 cells, suggesting a decreased BCR/Abl translation. The proteasome inhibitor MG132 or the pan-caspase inhibitor z-VAD-fmk extended BCR/Abl expression under oxygen/glucose shortage in K562 cells. Glucose shortage induced autophagy-dependent BCR/Abl protein degradation in KCL22 cells. Overall, our results showed that energy restriction induces different cell-specific BCR/Abl protein suppression patterns, which represent a converging route to TKi-resistance of CML cells. Thus, the interference with BCR/Abl expression in environment-adapted CML cells may become a useful implement to current therapy.

Synergistic effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway or NUP214-ABL1 fusion protein in human Acute Lymphoblastic Leukemia.

Philadelphia chromosome-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) accounts for 25-30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes are markers of poor prognosis and amplification of the NUP214-ABL1 oncogene can be detected mainly in patients with T-ALL. T cell malignancies harboring the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been achieved. The PI3K/Akt/mTOR signaling pathway is often activated in leukemias and plays a crucial role in leukemogenesis.We analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on three NUP214-ABL1 positive T-ALL cell lines that also displayed PI3K/Akt/mTOR activation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis. Four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226, ZSTK474 and Torin-2, showed marked cytotoxic effects on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of these compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL.

Dual transcripts of BCR-ABL & different polymorphisms in chronic myeloid leukaemia patients.

BACKGROUND & OBJECTIVES: Chronic myeloid leukaemia is (CML) characterized by the presence of a hallmark chromosomal translocation, the Philadelphia chromosome. Although there are many reports available regarding the different variants of BCR-ABL in CML, we studied the co-expression of e13a2 and e14a2 transcripts and a few polymorphisms in CML patients. METHODS: Molecular genetics approach was adapted to screen for polymorphisms, mutation and translocation in BCR, ABL kinase domain and BCR-ABL breakpoint region in 73 CML patients. RESULTS: All eight patients with dual transcripts were found to harbour an exonic polymorphism (c.2700 T>C) and an intronic polymorphism (g.109366A>G) that were earlier reported to be associated with co-expression of both the transcripts. We also observed c.763G>A mutation in ABL kinase domain and two polymorphisms, c.2387 A>G and c.2736A>G in the BCR gene. INTERPRETATION & CONCLUSIONS: Though our data support the previous findings that co-expression of BCR-ABL transcripts is due to the occurrence of exonic and intronic polymorphisms in the BCR gene, it also shows that the intronic polymorphism can arise without the linked exonic polymorphism. The occurrence of ABL kinase domain mutation is less frequent in Indian population.

Depression accelerates the development of gastric cancer through reactive oxygen species­activated ABL1 (Review).

Depression is a common symptom among gastric cancer (GC) patients and serves as a potential indication of poor prognosis and advanced cancer clinical stage. However, the molecular mechanism of depression­associated poor prognoses of GC patients remains unclear. Recent studies have revealed that GC patients with depression are under high levels of oxidative stress (OS) status that is accompanied by the dysfunction of numerous proto­oncogenes, including the ABL proto­oncogene 1 (ABL1), which is a non­receptor tyrosine kinase. Recent evidence indicates that ABL1 was dysregulated in both major depressive disorder (MDD) and cancer patients with depression, and high levels of reactive oxygen species (ROS) can lead to the activation of ABL1 in response to OS and that activated ABL1 subsequently contributes to development of GC via interactions with the downstream targets and corresponding signaling pathways. In this review, we examine the evidence to illuminate the molecular mechanism of ABL1 in the progression of GC patients with depression and identify out new and effective methods for the initial and long­term treatment of GC.

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.

The kinase ABL phosphorylates the microprocessor subunit DGCR8 to stimulate primary microRNA processing in response to DNA damage.

The DNA damage response network stimulates microRNA (miRNA) biogenesis to coordinate repair, cell cycle checkpoints, and apoptosis. The multistep process of miRNA biogenesis involves the cleavage of primary miRNAs by the microprocessor complex composed of the ribonuclease Drosha and the RNA binding protein DGCR8. We found that the tyrosine kinase ABL phosphorylated DGCR8, a modification that was required for the induction of a subset of miRNAs after DNA damage. Focusing on the miR-34 family, ABL stimulated the production of miR-34c, but not miR-34a, through Drosha/DGCR8-dependent processing of primary miR-34c (pri-miR-34c). This miRNA-selective effect of ABL required the sequences flanking the precursor miR-34c (pre-miR-34c) stem-loop. In pri-miRNA processing, DGCR8 binds the pre-miR stem-loop and recruits Drosha to the miRNA. RNA cross-linking assays showed that DGCR8 and Drosha interacted with pri-miR-34c, but we found an inverse correlation between ABL-stimulated processing and DGCR8 association with pri-miR-34c. When coexpressed in HEK293T cells, ABL phosphorylated DGCR8 at Tyr(267). Ectopic expression of a Y267F-DGCR8 mutant reduced the recruitment of Drosha to pri-miR-34c and prevented ABL or Drosha from stimulating the processing of pri-miR-34c. In mice engineered to express a nuclear import-defective mutant of ABL, miR-34c, but not miR-34a, expression was reduced in the kidney, and apoptosis of the renal epithelial cells was impaired in response to cisplatin. These results reveal a new pathway in the DNA damage response wherein ABL-dependent tyrosine phosphorylation of DGCR8 stimulates the processing of selective primary miRNAs.

Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding.

RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.

Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency.

BACKGROUND: The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID). OBJECTIVE: We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency. METHODS: We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of γH2AX accumulation was studied after ionizing radiation. RESULTS: Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect. CONCLUSIONS: Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.