ABL Tyrosine Kinase Plays an Important Role in Mechanisms Involved in Genomic Instability in Multiple Myeloma

Abstract Homologous recombination (HR) is a DNA repair mechanism that uses extensive sequence homology in the participating DNA molecules for an accurate repair. In a normal cellular environment, HR is the most precise DNA repair mechanism and therefore has a unique role in the maintenance of genomic integrity and stability. Normally HR is tightly regulated, however, as it involves incision and recombination of genomic DNA fragments, if dysregulated or dysfunctional, it can also be deleterious. Consistent with this view, we have shown that elevated HR activity mediates genomic instability and development of drug resistance in MM. Here we have now investigated the mechanism that may contribute to dysregulation of HR and genomic instability in MM, as well as evaluated an agent able to decrease acquisition of new genomic changes. It has been shown that Abl kinase regulates recombinase RAD51 by affecting its expression, stability as well as phosphorylation at Y315. Phosphorylation of RAD51 (at Y315) mediates its dissociation from BCR-ABL1 kinase and migration to the nucleus to form nuclear foci, one of the initial steps in HR. We have evaluated nilotinib, a small molecule inhibitor of Abl kinase and observed that it inhibited HR activity in all MM cell lines tested, in a dose-dependent manner. At 5 µM, nilotinib inhibited HR activity in MM1S, RPMI 8226 and U266 MM cells by 64%, 78% and 80%, respectively. Nilotinib led to reduced phosphorylation of RAD51 at Y315, the phosphorylation which affects RAD51 migration. Nilotinib-mediated inhibition of RAD51 and HR activity was also associated with reduced DNA breaks, as indicated by reduced levels of g-H2AX. To determine the impact of nilotinib on genome stability, MM (RPMI 8226) cells were cultured in the presence of nilotinib for three weeks and the impact of this treatment on appearance of new copy number changes was evaluated using SNP arrays. Using day 0 cells as baseline to identify new copy number events at 3 weeks, the acquisition of new genomic changes was inhibited by 50% in the presence of nilotinib. As we have previously reported that induction of HR helps develop dexamethasone resistance in a short period of time, we investigated whether inhibition of HR by nilotinib may improve efficacy of melphalan and dexamethasone in MM. Nilotinib (at 2.5 µM) significantly increased the efficacy of melphalan (10 µM); and dexamethasone (10 nM) in RPMI 8226 cells. The relation between these observed effects and inhibition of HR is being investigated. In conclusion, we have observed that Abl tyrosine kinase plays an important role in genomic instability in myeloma and its inhibition using nilotinib, suppresses the underlying mechanism of genomic instability and reduces acquisition of new genomic changes with potential for clinical application. Disclosures No relevant conflicts of interest to declare.

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Integrated Genomics and Functional Validation Identifies a Global Kinase Gene Signature Impacting Genome Stability in Myeloma

Blood ◽

10.1182/blood-2019-129069 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 363-363

Author(s):

Subodh Kumar ◽

Leutz Buon ◽

Srikanth Talluri ◽

Chengcheng Liao ◽

Jialan Shi ◽

...

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Genomic Instability ◽

Small Molecule ◽

Copy Number ◽

Genome Stability ◽

Dna Breaks ◽

Genomic Amplification ◽

Data Set ◽

The Impact

Identification of mechanisms underlying genomic instability is necessary to understand disease progression, including development of drug resistance. Our previous data demonstrates that dysregulation of DNA repair and maintenance/modification activities (including homologous recombination (HR), apurinic/apyrimidinic nuclease and APOBEC) significantly contribute to genomic instability in multiple myeloma (MM). However, how these and other pathways involved in genomic instability are dysregulated, remains to be explored. Since kinases play a critical role in the regulation of the maintenance of genomic integrity, we have performed a genome-wide kinome profiling to identify those involved in genomic instability in cancer. First, we analyzed genomic database for ten human cancers (including MM) from TCGA with both tumor cell gene expression and SNP/CGH array-based copy number information for each patient.We assessed genomic instability in each patient based on the total number of amplification and deletion events. We next interrogated all 550 kinases expressed in humans and identified those whose expression correlated with copy number alteration (based on FDR ≤ 0.05) in all tumor types. We identified six kinases whose elevated expression correlated with increased genomic instability defined by genomic amplification/deletion events in all ten cancers, including MM. To demonstrate functional relevance of these kinases, we conducted a CRISPR-based loss of function screen (using 3 guides per gene) in MM cells and evaluated the impact of each gene-knockout on micronuclei, a marker of ongoing genomic rearrangements and instability. For all six kinases, at least one guide resulted in ≥ 65% inhibition of micronuclei formation. Moreover, for five out of the six kinases, at least two guides showed ≥ 60% inhibition of micronuclei. All together, these data establishes a strong relevance of these kinases with genomic instability in MM. PDZ Binding Kinase (PBK) was among top kinases impacting genome stability in this data set with 2 out of 3 guides causing > 88% and 3rdguide causing 35% inhibition of micronuclei formation. We further report that inhibition of PBK, by knockdown or small molecule, inhibits DNA breaks, RAD51 recombinase expression and homologous recombination in MM cells. We further investigated molecular mechanisms involved in PBK-mediated genomic instability in MM. Expression profiling using RNA sequencing of MM cells treated with a specific PBK inhibitor showed that top ten pathways downregulated by treatment were mostly DNA repair/recombination followed by replication and G2/M checkpoint. Interestingly, we identified a notable overlap between PBK-regulated genes with FOXM1 target genes. FOXM1 is a major transcriptional regulator of genes involved in DNA repair, G2/M regulation and chromosomal stability. We, therefore, investigated PBK/FOXM1 interaction and show that PBK interacts with FOXM1 in MM cells. Moreover, the inhibition of PBK, by knockdown or small molecule, inhibits phosphorylation of FOXM1 as well as downregulates FOXM1-regulated HR and cell cycle genes RAD51, EXO1 and CDC25A. These results suggest that PBK-dependent phosphorylation of FOXM1 activity controls transcriptional networks involved in genomic instability in MM. Ongoing work is investigating role of PBK and other kinases in progression of MGUS/SMM to active MM and their impact on ongoing genomic changes with influence on multiple DNA repair pathways including HR. In conclusion, we describe a kinase panel that may have significant role in maintaining genome stability, and their perturbation may allow to improve genome stability in MM. Disclosures Munshi: Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Takeda: Consultancy.

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ABL1 Kinase Plays an Important Role in Spontaneous and Melphalan-Induced Genomic Instability in Multiple Myeloma: Potential Therapeutic Application

Blood ◽

10.1182/blood-2020-142793 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 51-51

Author(s):

Subodh Kumar ◽

Srikanth Talluri ◽

Jiangning Zhao ◽

Chengcheng Liao ◽

Lakshmi B. Potluri ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Genomic Instability ◽

Copy Number ◽

Critical Role ◽

Replication Stress ◽

Private Company ◽

Abl Kinase ◽

The Impact

Dysregulated homologous recombination (HR) contributes to the acquisition of genomic changes and development of drug resistance over time in multiple myeloma (MM). We have now further investigated the molecular intermediates that may play an important role in HR process. We observe that ABL kinase, which regulates RAD51 through its phosphorylation, contributes to increased HR activity and genomic instability in myeloma. Moreover, Cellular response after DSBs involves nuclear re-localization of ABL1 upon DNA damage. A cellular localization of ABL1 has been confirmed in MM. Consistently, we observe that ABL kinase inhibitor reduces HR activity and genomic instability (as assessed by micronucleus assay) in MM cells. Based on our observation that melphalan increases genomic instability (as assessed by micronuclei assay and by whole genome sequencing), we sought to investigate impact of melpahalan on HR and role of ABL1 kinase in this process. We show that treatment with melphalan leads to increase in RAD51 expression and HR activity in MM cells in a dose-dependent manner. Evaluation by RNA sequencing showed that treatment of MM1S cells with melphalan is associated with upregulation of p53 signaling (containing multiple genes involved in DNA damage, detection of damaged sites, recombination/repair and genomic instability) as the topmost pathway. These findings suggest that melphalan-induced DNA damage leads to a concerted overexpression of genes involved in DNA damage response, recombination, genomic instability and chemoresistance. With the role of ABL1-kinase following DSB, we investigated and report that treatment with melphalan induces micronuclei formation, whereas nilotinib significantly reduces both the basal and melphlalan-induced micronuclei in all MM cell lines tested. We have now confirmed these observations by evaluating copy number alterations using single nucleotide polymorphism (SNP) arrays. To evaluate the impact of nilotinib, melphalan and their combination on genomic instability, we cultured MM cells in the presence of nilotinib (2.5 µM), melphalan (1 µM) and combination of both drugs for three weeks and investigated the acquisition of new copy number events, relative to "day 0" cells (serving as baseline genome), using SNP arrays. Treatment with melphalan led to massive increase in the acquisition of amplification and deletion events, whereas nilotinib not only reduced the acquisition of copy number events under spontaneous condition but also almost completely reversed/prevented those induced by melphlalan. Importantly, the treatment with nilotinib could also significantly sensitized MM cell lines and bone marrow plasma cells from relapsed MM patients to melphalan treatment. These data confirm that ABL1 inhibition reduces spontaneous and melphalan-induced genomic instability in MM cells. Since components of cell cycle play critical role in the maintenance of genome stability and growth, we investigated the impact of ABL1-inhibitor nilotinib, alone and in combination with melphalan, on cell cycle. Treatment of MM1S cells with melphalan for 48hrs led to 2.5-fold increase in the accumulation of cells in S-phase, suggesting an increase in replication stress by melphalan. Nilotinib reduced both the spontaneous and melphalan-induced fraction of S-phase cells by 27% (±2%) and 30% (±1%), respectively. Combined treatment with nilotinib and melphalan also increased sub G1 fraction of cells by 2.3-fold compared to those treated with melphalan alone, suggesting increased apoptosis in these cells. These data are consistent with our observation that treatment of MM cells with melphalan increase the phosphorylation of RPA32, a marker of replication stress while nilotinib reduces both the endogenous and melphalan-induced phosphorylated RPA32 level, suggesting that nilotinib might also be helpful in combating replication stress-mediated genomic instability. Taken together, these data demonstrate the critical role of ABL1-kinase in both spontaneous and drug (melphalan)-induced genomic instability and its inhibition could reduce/delay genomic evolution while enhancing cytotoxicity in multiple myeloma. Disclosures Fulciniti: NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

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A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation

Blood ◽

10.1182/blood-2006-04-016113 ◽

2006 ◽

Vol 108 (5) ◽

pp. 1716-1723 ◽

Cited By ~ 45

Author(s):

Lynn McCallum ◽

Susan Price ◽

Nathalie Planque ◽

Bernard Perbal ◽

Andrew Pierce ◽

...

Keyword(s):

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Direct Consequence ◽

Suppressor Gene ◽

Hematopoietic Stem ◽

Protein Levels ◽

Abl Kinase ◽

Abl Tyrosine Kinase ◽

Ccn3 Expression

Chronic myeloid leukemia (CML) is characterized by the presence of the constitutively active BCR-ABL protein tyrosine kinase. Using a multipotent hemopoietic cell line, FDCP-Mix, expressing BCR-ABL tyrosine kinase, we investigated the initial effects of this kinase in primitive hematopoietic stem cells. We identified down-regulation of a novel gene, CCN3, as a direct consequence of BCR-ABL kinase activity. CCN3 has been reported to function as a tumor suppressor gene in solid tumors. Northern and Western blotting plus immunocytochemical analysis confirmed CCN3 expression is decreased and is tyrosine-phosphorylated in BCR-ABL kinase active FDCP-Mix cells. Decreased cellular CCN3 correlated with increased CCN3 secretion in BCR-ABL kinase active cells. In vitro treatment of human CML cell lines with imatinib or siRNA directed against BCR-ABL significantly reduced BCR-ABL while increasing CCN3 expression. Cells from patients responding to imatinib showed a similar decrease in BCR-ABL and increase in CCN3. CML CD34+ cells treated with imatinib in vitro demonstrated increased CCN3 protein. Transfecting CCN3 into BCR-ABL+ cells inhibited proliferation and decreased clonogenic potential. CCN3 plays an important role in internal and external cell-signaling pathways. Thus, BCR-ABL can regulate protein levels by governing secretion, a novel mechanism for this tyrosine kinase.

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Dysregulated Aid/Apobec Family Proteins Promote Genomic Instability in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.803.803 ◽

2016 ◽

Vol 128 (22) ◽

pp. 803-803

Author(s):

Srikanth Talluri ◽

Mehmet Kemal Samur ◽

Leutz Buon ◽

Stekla A Megan ◽

Purushothama Nanjappa ◽

...

Keyword(s):

Genomic Instability ◽

Cell Lines ◽

Copy Number ◽

Cytidine Deaminase ◽

Myeloma Cell ◽

Mrna Editing ◽

Genomic Changes ◽

Elevated Expression ◽

Apobec Family

Abstract The AID/APOBEC family of cytidine deaminase proteins includes AID (activity induced deaminase), and 10 related APOBEC enzymes (A1, A2, A3A, A3B, A3C, A3D, A3F, A3G, A3H and A4). AID has been well-studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes whereas APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Dysregulated activity of APOBECs causes C >T transitions or C>G, C>A transversions in DNA. We have recently shown APOBEC signature mutation pattern in multiple myeloma (MM) genomes (Bolli et al Nat. Comm. 2014), and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in myeloma. A role for the AID/APOBECs in generation of somatic mutations has also been proposed in a variety of other cancers based on identification of APOBEC signature mutations In order to understand which APOBECs are dysregulated in myeloma, we performed RNA sequencing analysis of primary myeloma cells from 409 newly-diagnosed MM patients and myeloma cell lines. Our analysis showed elevated expression of several APOBEC family members; mainly A3A, A3B, A3C, and A3G. We then optimized a plasmid-based functional assay and found high cytidine deaminase activity in extracts from a number of myeloma cell lines and patient derived CD138+ cells compared to CD138+ cells from healthy donors, suggesting that APOBECs are dysregulated in myeloma. We then investigated the impact of elevated APOBEC expression/function on overall genome maintenance and acquisition of genomic changes (such as amplifications, deletions) overtime. We used shRNA-mediated knockdown of specific APOBEC proteins in myeloma cell lines and investigated the acquisition of genomic changes in control and knockdown cells during their growth in culture, using SNP (Single Nucleotide Polymorphism) arrays and WGS (whole genome sequencing) platforms. Our results with both approaches showed significant reduction in the accumulation of copy number changes (both amplifications and deletions) and overall mutation load after APOBEC knockdown. Evaluation with both the SNP and WGS showed that when control and APOBEC knockdown cells were cultured for three weeks, the acquisition of new copy number and mutational changes throughout genome were reduced by ~50%. We next investigated the relationship between APOBEC expression/activity in MM and other DNA repair pathways. Using an in vitro HR activity assay, we measured HR activity in extracts from control and APOBEC knockdown cells. Depletion of APOBEC proteins resulted in 50-80% reduction in in vitro HR activity of the extracts. We also evaluated correlation between HR activity and gene expression using RNA-seq data from myeloma cells derived from 100 patients at diagnosis and identified the genes whose expression correlated with HR activity. Elevated expression of APOBECs 3D, 3G and 3F significantly correlated with high HR activity (R=0.3; P≤0.02), suggesting their relevance to HR. Analyzing genomic copy number information for each patient we have also observed significant correlation between higher expression of A3G and increased genomic instability in this dataset (P=0.0045). In summary, our study shows that dysregulated APOBECs induce mutations and genomic instability, and inhibiting APOBEC activity could reduce the rate of accumulation of ongoing genomic changes. This data sheds light on biology of the disease as well as clonal evolution. Disclosures Munshi: Amgen: Consultancy; Oncopep: Patents & Royalties; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Merck: Consultancy; Pfizer: Consultancy.

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Survivin silencing sensitizes imatinib-resistant CML cells to the cytotoxic effect of hydroxyurea

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.13119 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 13119-13119

Author(s):

F. Stagno ◽

E. Conte ◽

S. Stella ◽

E. Tirrò ◽

L. Manzella ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cell Lines ◽

Cytotoxic Effect ◽

Kinase Activity ◽

Point Mutations ◽

Survivin Expression ◽

Over Expression ◽

Abl Kinase ◽

Abl Tyrosine Kinase ◽

Apoptosis Protein

13119 Background: Imatinib Mesylate (IM), a semi-specific inhibitor of the BCR-ABL tyrosine kinase, is currently the treatment of choice for Chronic Myeloid Leukemia (CML). However, about one third of CML patients treated with IM develop resistance to the drug because of reactivation of BCR-ABL kinase activity. This phenomenon is usually ascribed to the amplification of the BCR-ABL gene or to the selection of leukemic clones harboring point mutations that abrogate IM binding. To identify novel anti-apoptotic signaling pathways employed by BCR-ABL and devise strategies capable of killing IM-resistant CML cells, we investigated the interplay between BCR-ABL and the Inhibitor of Apoptosis Protein Survivin. Methods: Murine hematopoietic cells (32D) transduced with p210 BCR-ABL and human cell lines either positive (K562, KCL22, KYO1 and LAMA84) or negative (HL60) for the BCR-ABL oncoprotein, were analyzed for Survivin expression by western blot before and after IM treatment. Three different pathways (MAPK, PI3K and JAK2/STA3) potentially involved in BCR-ABL-mediated induction of Survivin were studied using inhibitors specific for each signaling cascade. The effect of Survivin on the proliferation and viability of IM-sensitive and IM-resistant CML cells was investigated after silencing Survivin expression with small interfering RNAs. Results: BCR-ABL tyrosine kinase activity induced an over-expression of Survivin in both human and murine hematopoietic cell lines. This over-expression was both at the transcriptional and the translational level and required the JAK2/STAT3 pathway. Survivin silencing by siRNA increased IM cytotoxicity in IM-sensitive cells but failed to restore IM efficacy in IM-resistant cells. However, Survivin silencing sensitized CML cells to the cytotoxic effect of hydroxyurea and enhanced the efficacy of this compound on three different murine cell lines are insensitive to IM because of point mutations in the BCR-ABL kinase domain (Ba/F3p210Y253F, Ba/F3p210D276G and Ba/F3p210T315I). Conclusions: Reduction of Survivin expression improves the efficacy of IM and increases the sensitivity of IM-resistant CML cells to hydroxyurea. Survivin may represent an attractive therapeutic target for both IM-sensitive and IM-resistant CML patients. No significant financial relationships to disclose.

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Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.3116-3123.1985 ◽

1985 ◽

Vol 5 (11) ◽

pp. 3116-3123

Author(s):

J B Konopka ◽

O N Witte

Keyword(s):

Tyrosine Kinase ◽

Kinase Activity ◽

Myelogenous Leukemia ◽

Gene Products ◽

Tyrosine Kinase Activity ◽

Abl Kinase ◽

Abl Tyrosine Kinase ◽

Assay Conditions

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

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Decoupling of Normal CD40 / Interleukin-4 Immunoglobulin Heavy Chain Switch Signal Leads to Genomic Instability in RPMI 8226 and SGH-MM5 Multiple Myeloma Cell Lines.

Blood ◽

10.1182/blood.v104.11.1424.1424 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1424-1424

Author(s):

William Hwang ◽

Charles Gullo ◽

Gerrard Teoh

Keyword(s):

Dna Repair ◽

Cell Line ◽

Genomic Instability ◽

Cell Lines ◽

Heavy Chain ◽

Clonal Evolution ◽

Chromosomal Translocations ◽

Interleukin 4 ◽

Dna Dsbs ◽

Rpmi 8226

Abstract Certain genetic events accompany transformation of MM to more aggressive disease. For example, there is a predominance of new and promiscuous chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene on chromosome 14 (i.e. at 14q32). Development of chromosomal translocations involves the process of DNA double strand break repair (DSBR) by non-homologous end joining (NHEJ). DNA protein kinase (DNA-PK) is the principal DNA repair enzyme mediating DNA DSBR. It is made up of a catalytic subunit (DNA-PKcs) and a regulatory subunit (the Ku70/Ku86 heterodimer). Interestingly, the majority (86% to 100%) of freshly isolated patient MM cells express a variant form of Ku86 protein (Ku86v), which has been associated with abnormalities in DNA repair. Since, the combined effects of CD40 plus interleukin-4 (IL-4) are required for normal IgH isotype class switch recombination (CSR), and this process involves DNA DSBR, NHEJ and DNA-PK; we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormalities in DNA DSBR, which could lead eventually to genomic instability and clonal evolution. In this study, we first showed that RPMI 8226 and SGH-MM5 MM cell lines (but not the CESS Epstein-Barr virus (EBV) immortalized normal B cell line) that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger growth of tumor cell lines, suggesting that biological sequelae mediated by CD40 could be dysregulated in MM cells. Whether this process involves Ku86v is presently being investigated. We further demonstrate that CD40 triggering induced both DNA DSBs as well as new (acquired) karyotypic abnormalities in the SGH-MM5 MM cell line. These complex karyotypic changes included at least 5 new and clonal chromosomal translocations and deletions. Since, normal IgH isotype CSR is accompanied by induction of activation induced cytidine deaminase (AID) expression, we next demonstrated that CD40 triggering of MM cell lines (without IL-4) was sufficient to upregulate AID expression. These data suggest that DNA DSBs induced by CD40 were part of IgH isotype CSR rather than CD40 induced apoptosis of tumor cells. This is an important distinction to make because CD40 has been demonstrated to induce apoptosis by both p53 -dependent and -independent pathways. In order to confirm that CD40-triggered MM cells did not undergo apoptosis, we performed annexin V/propidium iodide (PI) staining on CD40-triggered MM cells. We showed that MM cell lines not only remained viable after CD40 triggering, but also demonstrated G1 cell cycle exit. In conclusion, our present study shows that CD40 alone could act as an inducer of genomic instability in MM cell lines, and lead to clonal evolution. Since CD40 ligand (CD40L) is naturally expressed during inflammation and T cell activation, it is tempting to speculate that the normal inflammatory process could potentially participate in clonal evolution and even myelomagenesis in vivo.

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Rewiring of the Protein Kinase C Beta 2 (PKC βII) and Bcr/Abl Signal Transduction Pathways

Blood ◽

10.1182/blood.v118.21.1673.1673 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1673-1673

Author(s):

David Arthur Hoekstra ◽

Louise M Carlson ◽

Kelvin P Lee

Keyword(s):

Signal Transduction ◽

Plasma Membrane ◽

Tyrosine Kinase ◽

Growth Arrest ◽

K562 Cells ◽

Signal Transduction Pathways ◽

Abl Kinase ◽

Abl Tyrosine Kinase ◽

Induction Of Apoptosis ◽

Constitutively Active

Abstract Abstract 1673 Chronic myeloid leukemia (CML) accounts for 20% of adult leukemias, and is characterized by the presence of the bcr-abl fusion gene. This gene leads to the production of a constitutively active Abl tyrosine kinase, which promiscuously phosphorylates/activates a variety of intracellular signal transduction pathways. The presence of constitutively active Abl is essential for CML blast survival even in advanced disease, and underlies the success that the Bcr/Abl kinase inhibitor imatinib has had. However, resistance to imatinib occurs in a significant number of accelerated phase or blast crisis patients and is a significant clinical obstacle. Instead of trying to inhibit Bcr/Abl signaling, we propose a previously unexplored approach to ‘rewire’ kinase signaling pathways to activate a “suicide” prodrug that would not normally be activated by Bcr/Abl. We have previously shown that direct activation of PKCβII by Phorbol 12-myristate 13-acetate (PMA) drives dendritic cell differentiation in both normal and leukemic progenitors, as well as induces apoptosis and growth arrest. PKCβII is a member of the PKC family of serine/threonine kinases and is kept in an inactive state in the cytosol by interactions between its pseudosubstrate and kinase domains; upon activation, the pseudosubstrate domain releases the kinase domain, and PKCβII translocates to the plasma membrane. Substitution of an alanine at position 25 in the pseudosubstrate domain for a phosphomimetic glutamic acid leads to the constitutive activation of PKCβII. Similarly, we hypothesized that substituting the alanine at position 25 for a phosphorylatable tyrosine (A25Y) along with the corresponding Bcr/Abl kinase target motif (Ala-X-X-Ile-Tyr-X-X-Phe/Pro) into the pseudosubstrate domain of PKCβII, would allow the Bcr/Abl tyrosine kinase to activate the PKCβII signaling pathway. Bcr/Abl mediated activation of PKCβII would then lead to the induction of apoptosis, growth inhibition, and differentiation. Using confocal microscopy, we show that following transfection WT-PKCβII is cytoplasmically located in media alone and addition of PMA leads to translocation to the plasma membrane, indicating activation in both Bcr/Abl+ K562 cells, and Bcr/Abl− KG1a cells. However when A25Y-PKCβII constructs are transfected in, A25Y-PKCβII is found at the plasma membrane in K562, but not in KG1a cells in media alone. These observations were then quantified using ImageStream technology, which allows for simultaneous acquisition of both flow cytometric data, and high resolution fluorescent images. Using this technology, we show that A25Y-PKCβII is activated in media alone in K562 cells, and only upon addition of PMA in KG1a cells. Additionally, when Bcr/Abl was stably transfected in KG1a cells, A25Y-PKCβII was able to translocate to the plasma membrane in media alone, indicating activation by Bcr/Abl. Upon activation and translocation to the plasma membrane, PKCβII is rapidly degraded; accordingly, we show that expression of WT PKCβII decreases only by 20% over 72 hours post transfection, whereas expression of A25Y-PKCβII results in an average decrease of 90% over the same 72 hour time course. To test whether activation of A25Y-PKCβII leads to apoptosis and growth arrest, Bcr/Abl+ K562, and Bcr/Abl− KG1a cells were transfected with either WT and A25Y-PKCβII and measured for apoptosis with AnnexinV using Flow Cytometry. We found that A25Y-PKCβII induced a maximum of a 4-fold increase of apoptosis when compared to WT PKCβII in K562 cells; however there was no increase observed in KG1a cells. This work demonstrates that rewiring PKCβII to be inducible by Bcr/Abl is feasible, and that activation of PKCβII by Bcr/Abl induces characteristic translocation to the plasma membrane, and induction of apoptosis. Future work will address whether induction of DC differentiation is maintained in Bcr/Abl activated PKCβII cells, as well as the molecular kinetics of this activation. Disclosures: No relevant conflicts of interest to declare.

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The Impact Of Novel Splicing Abnormalities Of BCR-ABL On The Pathogenesis Of CML

Blood ◽

10.1182/blood.v122.21.5159.5159 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5159-5159

Author(s):

Junichiro Yuda ◽

Toshihiro Miyamoto ◽

Yoshikane Kikushige ◽

Jun Odawara ◽

Yasuyuki Ohkawa ◽

...

Keyword(s):

Tyrosine Kinase ◽

Conflicts Of Interest ◽

Kinase Domain ◽

Molecular Response ◽

Healthy Individuals ◽

Wild Type ◽

Abl Kinase ◽

Tki Treatment ◽

The Impact ◽

Splicing Patterns

Abstract Background Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain point mutations. The mechanism of resistance in patients without BCR-ABL kinase domain point mutation is still elusive. Previous studies have revealed the abnormal splicing of BCR-ABL kinase domain, including Exon8/9 junction 35bp insertion and exon-skipping of Exon 7 (T O'Hare et al, Blood 2011. Gaillard JB, et al. Mol Cancer Ther 2010). The insertion of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues and inactive tyrosine kinase activity. The effect of these splicing abnormalities on susceptibility of cells against TKIs is still controversial. Furthermore, the conventional direct sequence techniques could not evaluate splicing abnormalities in major molecular response (MMR)- complete molecular response (CMR) CML patients, who achieved clinically leukemia-free state with a small number residual CML stem cells. Aims The aim of this study is to evaluate the frequency and the patterns of splicing abnormalities of BCR-ABL in CML patients, especially who achieved MMR-CMR by TKI treatment. Methods We analyzed peripheral blood samples from healthy individuals and CML chronic phase patients. We extracted total RNA from these samples and synthesized cDNA, and then performed PCR-amplification of BCR-ABL kinase domain in CML patients and ABL kinase domain in healthy individuals, respectively. PCR products were subjected to the amplicon sequence: We deeply sequenced BCR-ABL fusion gene transcripts, and evaluated splicing forms of BCR-ABL by using HiSeq 2000 (illumina). Results We successfully established a novel analysis method, which can detect the pattern of splicing abnormalities even in MMR-CMR patients. Using the amplicon sequence technique, we detected abnormal splicing patterns of BCR-ABL in 5 out of 15 CML patients. We also found that the splicing abnormalities were not restricted to 35bp insertion at the exon8/9 junction, thus intronic retention of intron 8 and intron 9 could be frequently detected with or without the 35bp insertion in CML patients (Table 1). Of note, these abnormal splicing patterns always co-existed with wild type BCR-ABL transcripts in all 5 cases analyzed. In addition to the novel splicing abnormalities in CML, we unexpectedly found in healthy individuals that splicing abnormalities such as 35bp insertion at the exon8/9 junction and intronic retention could be detected in ABL1 transcripts (Table 1). This result suggests that this sort of splicing abnormalities could occur at a certain frequency in steady state human hematpoiesis, and is not specific to BCR-ABL. Summary / Conclusion We have newly established an analysis system to efficiently detect splicing abnormalities of BCR-ABL even in MMR-CMR CML patients. Using this highly efficient amplicon sequence technique, we identified novel splicing abnormalities both in healthy individuals and CML patients, and found that the wild type BCR-ABL transcripts always co-exist with abnormally spliced BCR-ABL transcripts. These results collectively suggest that splicing abnormalities within the ABL1 kinase domain are not specific to CML patients treated with TKIs, and that the detection of such kinase domain splicing abnormalities do not reflect insusceptibility of the remaining cells during TKI treatment. Disclosures: No relevant conflicts of interest to declare.

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