Real-world treatment patterns and outcomes for patients with multiple myeloma in Denmark, Finland and Sweden: An analysis using linked Nordic registries.

AIM: The Health outcomes and Understanding of MyelomA multi-National Study (HUMANS) was a large-scale, retrospective study conducted across Denmark, Finland and Sweden using linked data from national registries. We describe the characteristics, treatment patterns and clinical outcomes for patients with newly diagnosed multiple myeloma (NDMM) over 2010-2018. METHODS: Patients with NDMM who received MM-specific, first-line treatments, were categorised by treatment (autologous stem cell transplantation [ASCT] or a combination chemotherapy regimen based on bortezomib, lenalidomide or melphalan-prednisolone-thalidomide). RESULTS: 11,023 patients received treatment over 2010-2018. Time between diagnosis and treatment was shortest in Denmark (0.9 months), then Sweden (2.9 months) and Finland (4.6 months). Around one third of patients underwent ASCT. Lenalidomide-based regimens were prescribed to 23-28% of patients in Denmark and Finland, versus 12% in Sweden. Patients receiving lenalidomide had the longest wait for treatment, from 3.2 months (Denmark) to 12.1 months (Sweden). Treatment persistence was highest among patients receiving melphalan-prednisolone-thalidomide (7-8 months) in Finland and Sweden and lowest among those receiving bortezomib (3.5 months) in Finland. Overall survival (OS) was longest among patients with ASCT (7-10 years). Among patients receiving chemotherapy, OS (from diagnosis/treatment initiation), varied between cohorts. In a sensitivity analysis excluding patients with smouldering MM, OS decreased for all; for patients receiving bortezomib or lenalidomide, OS from diagnosis was 40-49 and 27-54 months, respectively. CONCLUSIONS: This population-based study of patients with NDMM receiving first-line MM-specific treatment, provides real-world data on treatment patterns and outcomes to complement data from randomised clinical trials.

Use of Linked Nordic Registries for Population Studies in Hematologic Cancers: The Case of Multiple Myeloma.

Purpose: Linked health-care registries and high coverage in Nordic countries lend themselves well to epidemiologic research. Given its relatively high incidence in Western Europe, complexity in diagnosis, and challenges in registration, multiple myeloma (MM) was selected to compare registries in Denmark, Finland, and Sweden. Patients and Methods: Data were obtained from four archetypal registries in each country (spanning January 2005-October 2018): National Patient Registry (NPR), Prescribed Drug Registry (PDR), Cancer Registry (CR), and Cause of Death Registry. Patients newly diagnosed with MM who received MM-specific treatment were included. PDR/NPR treatment records were used to assess incident NPR cases. The registration quality of MM-specific drugs in the PDR of each country was also evaluated. Results: In Denmark, only 6% of patients in the NPR were not registered in the CR; in Sweden, it was 16.9%. No systematic differences were identified that could explain this discrepancy. In Denmark, lenalidomide and bortezomib were registered in the NPR with high coverage, but less expensive drugs typically given in combination with bortezomib were not covered in any of the registries. In Finland and Sweden, bortezomib records were not identified in the PDR, but some were in the NPR; other drugs had good coverage in the PDR. Conclusions: The registries evaluated in this study can be used to identify the MM population; however, given the gaps in MM registration in the Finnish and Swedish CRs, Danish registries provide the most comprehensive datasets for research on treatment patterns for MM.

Protein kinase A type I activates a CRE-element more efficiently than protein kinase A type II regardless of C subunit isoform.

BACKGROUND: Protein kinase A type I (PKAI) and PKAII are expressed in most of the eukaryotic cells examined. PKA is a major receptor for cAMP and specificity is achieved partly through tissue-dependent expression and subcellular localization of subunits with different biochemical properties. In addition posttranslational modifications help fine tune PKA activity, distribution and interaction in the cell. In spite of this the functional significance of two forms of PKA in one cell has not been fully determined. Here we have tested the ability of PKAI and PKAII formed by expression of the regulatory (R) subunits RIα or RIIα in conjunction with Cα1 or Cß2 to activate a co-transfected luciferace reporter gene, controlled by the cyclic AMP responsive element-binding protein (CREB) in vivo. RESULTS: We show that PKAI when expressed at equal levels as PKAII was significantly (p < 0.01) more efficient in inducing Cre-luciferace activity at saturating concentrations of cAMP. This result was obtained regardless of catalytic subunit identity. CONCLUSION: We suggest that differential effects of PKAI and PKAII in inducing Cre-luciferace activity depend on R and not C subunit identity.

MEK1 and MEK2 regulate distinct functions by sorting ERK2 to different intracellular compartments.

In this study, we provide novel insight into the mechanism of how ERK2 can be sorted to different intracellular compartments and thereby mediate different responses. MEK1-activated ERK2 accumulated in the nucleus and induced proliferation. Conversely, MEK2-activated ERK2 was retained in the cytoplasm and allowed survival. Localization was a determinant for ERK2 functions since MEK1 switched from providing proliferation to be a mediator of survival when ERK2 was routed to the cytoplasm by the attachment of a nuclear export site. MEK1-mediated ERK2 nuclear translocation and proliferation were shown to depend on phosphorylation of S298 and T292 sites in the MEK1 proline-rich domain. These sites are phosphorylated on cellular adhesion in MEK1 but not MEK2. Whereas p21-activated kinase phosphorylates S298 and thus enhances the MEK1-ERK2 association, ERK2 phosphorylates T292, leading to release of active ERK2 from MEK1. On the basis of these results, we propose that the requirement of adhesion for cells to proliferate in response to growth factors, in part, may be explained by the MEK1 S298/T292 control of ERK2 nuclear translocation. In addition, we suggest that ERK2 intracellular localization determines whether growth factors mediate proliferation or survival and that the sorting occurs in an adhesion-dependent manner.

Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit.

BACKGROUND: Epidermal Growth Factor Receptor (EGFR) is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA) induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits. RESULTS: We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Calpha and Cbeta in two different models. The first model used targeted disruption of either Calpha or Cbeta in mice whereas the second model used Calpha and Cbeta RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level. CONCLUSION: Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.

Inactive forms of the catalytic subunit of protein kinase A are expressed in the brain of higher primates.

It is well documented that the beta-gene of the catalytic (C) subunit of protein kinase A encodes a number of splice variants. These splice variants are equipped with a variable N-terminal end encoded by alternative use of several exons located 5' to exon 2 in the human, bovine and mouse Cbeta gene. In the present study, we demonstrate the expression of six novel human Cbeta mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CbetaDelta4, were identified in low amounts at the mRNA level in NTera2-N cells. We developed a method to detect CbetaDelta4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CbetaDelta4 variants demonstrated that they are catalytically inactive both in vitro against typical protein kinase A substrates such as kemptide and histone, and in vivo against the cAMP-responsive element binding protein. Furthermore, co-expression of CbetaDelta4 with the regulatory subunit (R) followed by kinase activity assay with increasing concentrations of cAMP and immunoprecipitation with extensive washes with cAMP (1 mm) and immunoblotting demonstrated that the CbetaDelta4 variants associate with both RI and RII in a cAMP-independent fashion. Expression of inactive C subunits which associate irreversibly with R may imply that CbetaDelta4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.

Protein kinase A associates with HA95 and affects transcriptional coactivation by Epstein-Barr virus nuclear proteins.

HA95, a nuclear protein homologous to AKAP95, has been identified in immune precipitates of the Epstein-Barr virus (EBV) coactivating nuclear protein EBNA-LP from EBV-transformed lymphoblastoid cells (LCLs). We now find that HA95 and EBNA-LP are highly associated in LCLs and in B-lymphoma cells where EBNA-LP is expressed by gene transfer. Binding was also evident in yeast two-hybrid assays. HA95 binds to the EBNA-LP repeat domain that is the principal coactivator of transcription. EBNA-LP localizes with HA95 and causes HA95 to partially relocalize with EBNA-LP in promyelocytic leukemia nuclear bodies. Protein kinase A catalytic subunit alpha (PKAcsalpha) is significantly associated with HA95 in the presence or absence of EBNA-LP. Although EBNA-LP is not a PKA substrate, HA95 or PKAcsalpha expression in B lymphoblasts specifically down-regulates the strong coactivating effects of EBNA-LP. The inhibitory effects of PKAcsalpha are reversed by coexpression of protein kinase inhibitor. PKAcsalpha also inhibits EBNA-LP coactivation with the EBNA-2 acidic domain fused to the Gal4 DNA binding domain. Furthermore, EBNA-LP- and EBNA-2-induced expression of the EBV oncogene, LMP1, is down-regulated by PKAcsalpha or HA95 expression in EBV-infected lymphoblasts. These experiments indicate that HA95 and EBNA-LP localize PKAcsalpha at nuclear sites where it can affect transcription from specific promoters. The role of HA95 as a scaffold for transcriptional regulation is discussed.

Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cbeta2.

BACKGROUND: Two main genes encoding the catalytic subunits Calpha and Cbeta of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cbeta genes encode several splice variants, including the splice variant Cbeta2. In mouse Cbeta2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cbeta2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues. RESULTS: We identified a novel 47 kDa splice variant encoded by the mouse Cbeta gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cbeta2. Based on the present results and the existence of a human brain-specifically expressed Cbeta splice variant designated Cbeta4 that is identical to the former mouse Cbeta2 splice variant, the mouse splice variant has now been renamed mouse Cbeta4. CONCLUSION: Murine lymphoid tissues express a protein that is a homologue of human and bovine Cbeta2. The murine Cbeta gene encodes the splice variants Cbeta1, Cbeta2, Cbeta3 and Cbeta4, as is the case with the human Cbeta gene.

Identification and characterization of novel PKA holoenzymes in human T lymphocytes.

Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T-cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T-cell RNA using C subunit-specific primers revealed expression of two catalytically active PKA C subunits C alpha1 (40 kDa) and C beta2 (47 kDa) in these cells. Anti-RI alpha and Anti-RII alpha immunoprecipitations demonstrated that both C alpha1 and C beta2 associate with RI alpha and RII alpha to form PKAI and PKAII holoenzymes. Moreover, Anti-C beta2 immunoprecipitation revealed that C alpha1 coimmunoprecipitates with C beta2. Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme, released C alpha1 but not C beta2 from the Anti-C beta2 precipitate, indicating that C beta2 and C alpha1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP-responsive enzymes that may mediate specific effects of cAMP.

Protein kinase A (PKA)--a potential target for therapeutic intervention of dysfunctional immune cells.

In several cases of immunodeficiency and autoimmunity, the dysfunctional immune system is associated with either hypo- or hyperactive T and B cells. In autoimmune conditions such as systemic lupus erythematosus (SLE) and immunodeficiencies such as acquired immunodeficiency syndrome (AIDS), it has been demonstrated that the regulatory effect of the signaling pathway of cyclic 3', 5' adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) is abrogated. PKA is well-known as a key regulator of immune responses in that it inhibits both early and late phases of antigen induced T and B cell activation. Here we will discuss a potential useful strategy for therapeutic interventions of dysfunctional T cells associated with SLE and HIV by modulation of the cAMP-PKA pathway. Therefore, we will describe the components and architecture of the cAMP-PKA signaling pathway in T cells in order to point out one or several steps which potentially may serve as targets for therapeutic intervention.

Induction of Cbeta splice variants and formation of novel forms of protein kinase A type II holoenzymes during retinoic acid-induced differentiation of human NT2 cells.

Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are critical regulators of neuronal differentiation. The expression, levels and activities of PKA subunits were studied prior to and during differentiation of the human neuronal precursor cell line NTera 2 (NT2). Undifferentiated NT2 cells expressed mainly cytoplasmic PKA type I, consisting of the regulatory subunit RIalpha and the catalytic subunit Calpha. Low levels of PKA type II consisting of RIIalpha or RIIbeta associated with Calpha were also detected, mainly in the cytoplasm and in the Golgi-centrosomal area. During retinoic acid-induced differentiation, the RIalpha and RIIalpha expressions remained in the cytoplasm, while we observed a strong upregulation of RIIbeta, located to the whole cytoplasm including neurite extensions. This upregulation coincided with increased PKA-specific activity accompanied by a strong induction of a number of neuronal-specific Cbeta splice variants that together with RIIbeta form novel PKAII holoenzymes. Formation of novel PKAII holoenzymes may imply specific PKA features which may have consequences for the process of neuronal differentiation and nerve cell function.

Isoform-specific regulation of immune cell reactivity by the catalytic subunit of protein kinase A (PKA).

There are two major genes encoding the catalytic subunits of protein kinase A, Calpha and Cbeta. The functional significance of these isoforms is enigmatic. Lymphoid cells of the immune system express both Calpha and Cbeta. In this study we tested the role of Calpha and Cbeta in regulating immune cell reactivity to antigens using mice carrying a targeted disruption of the Calpha and Cbeta gene respectively. Calpha and Cbeta ablation both resulted in a 50% reduction in PKA-specific kinase activity and the level of PKA type I but not PKA type II. Moreover, despite that C subunit ablation did not affect immune cell development and homeostasis, Calpha but not Cbeta ablation augmented expression of the activation marker CD69 on lymphocytes. CD69 induction coincided with immune cell hyperresponsiveness and was associated with reduced sensitivity to cAMP-mediated inhibition of anti-CD3 induced T cell proliferation. Our results imply that Calpha is required for normal immune cell reactivity and demonstrates isoform-specific effects and non-redundant functions of C subunit isoforms expressed in the same cell.

Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing.

Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Calpha and Cbeta are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism.