cAMP:n säätelemä fosfoproteiini 19 toiminta akuutissa myelooisessa leukemiassa

Akuutti myelooinen leukemia (AML) on aggressiivinen veri- ja luuydinsyöpä. AML jaetaan useisiin alaryhmiin syöpäsolujen ilmiasun, erilaistumisasteen ja sytogenetiikan mukaan. Todellinen tämänhetkinen hoidollinen haaste AML:n taudinkuvassa on taudin uusiutuminen eli relapsi standardihoitojen jälkeen. Proteiinifosfataasi 2A (PP2A) on kasvunestäjäproteiini, jonka toiminnan estämisen on osoitettu olevan yksi syöpäsolujen syntymisen edellytyksistä. PP2A:n esto syöpäsoluissa tapahtuu useimmiten erityisten PP2A:n aktiivisuutta estävien inhibiittoriproteiinien toimesta. Yksi näistä on cAMP:n säätelemä fosfoproteiini 19 (ARPP19), ja sen on erityisesti osoitettu edistävän solujakautumista eli mitoosia. Aikaisemmissa tutkimuksissa on havaittu, että diagnoosivaiheen korkeampi ARPP19 ilmentymistaso ennustaa AML-potilaan taudin uusiutumista. Tästä jatkona, erikoistyössäni pyrin selvittämään ARPP19 molekyylitason toimintaa leukemiasoluissa. Tutkimuksessa käytettiin neljää keskenään ilmiasun, erilaistumisasteen ja sytogenetiikan mukaan mahdollisimman erilaista AML-solulinjaa. ARPP19 ilmentyminen solulinjoissa estettiin lentivirus transduktoiduilla shRNA:lla sekä Nucleofector™ teknologian avulla siirrostetuilla siRNA:lla. Western blottauksella todennettiin AML-solulinjojen ARPP19 hiljentyminen ja sen vaikutukset muiden proteiinien ilmentymistasoihin. ARPP19 hiljennetyn solulinjan aineenvaihdunnallista aktiivisuutta seurattiin luminesenssiin perustuvalla CellTiter-Glo® -menetelmällä, jota käytettiin myös tutkittaessa AML-solulinjojen vastetta PP2A:n reaktivaattori molekyyleihin (SMAPs) ja Greatwall kinaasi inhibiittori 1 (GKI-1), sekä kahdeksaan, anagrelidini, tretinoiini, tacrolimus, abirateroni, lenalidomidi, dasatinibi, selumetinibi ja XAV-939, muuhun lääkeaineeseen. Tehdyissä tutkimuksissa todettiin ARPP19 vaikuttavan AML-solulinjojen elinkykyisyyteen. Kun ARPP19 toimintaa tarkasteltiin proteiinitasolla, havaittiin ARPP19 vaikuttavan c-MYC:n, CIP2A:n ja Greatwall-kinaasin toimintaan, mikä pystyttiin näkemään sekä ARPP19 akuutisti, että pitkäaikaisesti poistetuista solulinjoista. Lisäksi joissakin tapauksissa ARPP19 havaittiin vaikuttavan Cdk1-kinaasin toimintaan. ARPP19 proteiinin toiminnan merkitystä AML-soluissa pystyttiin lopulta tarkentamaan, kun ARPP19 ilmentymistason osoitettiin vaikuttavan AML:n solujen lääkeainevasteeseen dasatinibiä kohtaan. Solulinjat, joiden ARPP19-proteiinitason ilmentyminen oli korkea, olivat vastustuskykyisiä annettavalle lääkeaineelle

Arpp19 Promotes Myc and Cip2a Expression and Associates with Patient Relapse in Acute Myeloid Leukemia

Disease relapse from standard chemotherapy in acute myeloid leukemia (AML) is poorly understood. The importance of protein phosphatase 2A (PP2A) as an AML tumor suppressor is emerging. Therefore, here, we examined the potential role of endogenous PP2A inhibitor proteins as biomarkers predicting AML relapse in a standard patient population by using three independent patient materials: cohort1 (n = 80), cohort2 (n = 48) and The Cancer Genome Atlas Acute Myeloid Leukemia (TCGA LAML) dataset (n = 160). Out of the examined PP2A inhibitors (CIP2A, SET, PME1, ARPP19 and TIPRL), expression of ARPP19 mRNA was found to be independent of the current AML risk classification. Functionally, ARPP19 promoted AML cell viability and expression of oncoproteins MYC, CDK1, and CIP2A. Clinically, ARPP19 mRNA expression was significantly lower at diagnosis (p = 0.035) in patients whose disease did not relapse after standard chemotherapy. ARPP19 was an independent predictor for relapse both in univariable (p = 0.007) and in multivariable analyses (p = 0.0001) and gave additive information to EVI1 expression and risk group status (additive effect, p = 0.005). Low ARPP19 expression was also associated with better patient outcome in the TCGA LAML cohort (p = 0.019). In addition, in matched patient samples from diagnosis, remission and relapse phases, ARPP19 expression was associated with disease activity (p = 0.034), indicating its potential usefulness as a minimal residual disease (MRD) marker. Together, these data demonstrate the oncogenic function of ARPP19 in AML and its risk group independent role in predicting AML patient relapse tendency

Discovery of a Novel CIP2A Variant (NOCIVA) with Clinical Relevance in Predicting TKI Resistance in Myeloid Leukemias

From Crossref journal articles via Jisc Publications RouterHistory: epub 2021-03-05, issued 2021-03-05, ppub 2021-05-15Publication status: PublishedFunder: Sigrid Juselius FoundationFunder: Turku Doctoral Program of Molecular MedicineFunder: Turku University Hospital ERVA; Grant(s): 13283, 13336Funder: Päivikki and Sakari Sohlbergin Foundation; FundRef: 10.13039/501100004212Funder: Cancer Foundation Väre; FundRef: 10.13039/100010128Funder: Finnish Cultural Foundation; FundRef: 10.13039/501100003125Abstract Purpose: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that inhibits the tumor suppressor PP2A-B56α. However, CIP2A mRNA variants remain uncharacterized. Here, we report the discovery of a CIP2A splicing variant, novel CIP2A variant (NOCIVA). Experimental Design: Characterization of CIP2A variants was performed by both 3′ and 5′ rapid amplification of cDNA ends from cancer cells. The function of NOCIVA was assessed by structural and molecular biology approaches. Its clinical relevance was studied in an acute myeloid leukemia (AML) patient cohort and two independent chronic myeloid leukemia (CML) cohorts. Results: NOCIVA contains CIP2A exons 1 to 13 fused to 349 nucleotides from CIP2A intron 13. Intriguingly, the first 39 nucleotides of the NOCIVA-specific sequence are in the coding frame with exon 13 of CIP2A and code for a 13-amino acid peptide tail nonhomologous to any known human protein sequence. Therefore, NOCIVA translates to a unique human protein. NOCIVA retains the capacity to bind to B56α, but, whereas CIP2A is predominantly a cytoplasmic protein, NOCIVA translocates to the nucleus. Indicative of prevalent alternative splicing from CIP2A to NOCIVA in myeloid malignancies, AML and CML patient samples overexpress NOCIVA, but not CIP2A mRNA. In AML, a high NOCIVA/CIP2A mRNA expression ratio is a marker for adverse overall survival. In CML, high NOCIVA expression is associated with inferior event-free survival among imatinib-treated patients, but not among patients treated with dasatinib or nilotinib. Conclusions: We discovered a novel variant of the oncoprotein CIP2A and its clinical relevance in predicting tyrosine kinase inhibitor therapy resistance in myeloid leukemias

PP2A inhibition is a druggable MEK inhibitor resistance mechanism in KRAS-mutant lung cancer cells

Kinase inhibitor resistance constitutes a major unresolved clinical challenge in cancer. Furthermore, the role of serine/threonine phosphatase deregulation as a potential cause for resistance to kinase inhibitors has not been thoroughly addressed. We characterize protein phosphatase 2A (PP2A) activity as a global determinant of KRAS-mutant lung cancer cell resistance across a library of >200 kinase inhibitors. The results show that PP2A activity modulation alters cancer cell sensitivities to a large number of kinase inhibitors. Specifically, PP2A inhibition ablated mitogen-activated protein kinase kinase (MEK) inhibitor response through the collateral activation of AKT/mammalian target of rapamycin (mTOR) signaling. Combination of mTOR and MEK inhibitors induced cytotoxicity in PP2A-inhibited cells, but even this drug combination could not abrogate MYC up-regulation in PP2A-inhibited cells. Treatment with an orally bioavailable small-molecule activator of PP2A DT-061, in combination with the MEK inhibitor AZD6244, resulted in suppression of both p-AKT and MYC, as well as tumor regression in two KRAS-driven lung cancer mouse models. DT-061 therapy also abrogated MYC-driven tumorigenesis. These data demonstrate that PP2A deregulation drives MEK inhibitor resistance in KRAS-mutant cells. These results emphasize the need for better understanding of phosphatases as key modulators of cancer therapy responses

Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1

Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies