Angiotensin II Receptor-Associated Protein (AGTRAP) Synergizes with Mpl Signaling to Promote Survival and to Increase Proliferation Rate of Hematopoietic Cells.

Abstract

3606 Poster Board III-542 The Mpl proto-oncogene plays a basic role in megakaryocytic development and platelet production as well as performs substantial function in hematopoietic stem cell (HSC) homeostasis and self-renewal. Several, mutations in Mpl have been shown to be associated with the myeloproliferative disorders. In an attempt to identify cellular factors that could cooperate with Mpl signaling leading to increases in cell survival and proliferation, we performed retroviral insertional mutagenesis using MSCV based vector coding for a drug dependent, dimerizable, fusion protein which contains the cytoplasmic domain of Mpl. Such vector construct can interrupt gene structure through insertion, and has an intact long terminal repeats which can activate adjacent genes. We used that vector construct to transduce the human leukemia cell line K562. After transduction, the endogenous transforming BCR-ABL kinase was blocked using Imatinib, and cells dependent on Mpl signaling were selected by the addition of dimerizing drug, AP20187. In the absence of Mpl signaling the cells underwent erythroid differentiation and died. Cells that acquired proliferative advantage and were dependent on Mpl function were expected to point to mutations synergistic with Mpl signaling. Cloning of retroviral integration sites (RIS) from the selected populations would allow to identify overrepresented clones. Indeed, overrepresented RIS clones were found in several independent transductions. Such overrepresented clones were not observed in an independent experiment where RIS were cloned from cells transduced with the same vector and sorted but not selected on Imatinib and AP20187. RIS from the overrepresented clones pointed to candidate Mpl cooperating genes. AGTRAP a member of renin-angiotensin system (RAS) was identified among the candidate genes. Multiple lines of evidence implicate RAS in hematopoietic cell proliferation and differentiation. RAS components, including renin, angiotensin-converting enzyme(ACE), angiotensin II, angiotensin type 1 and type 2 receptors (AT1 andAT2), and tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP an inhibitory hematopoietic peptide) have been isolated in the bone marrow as well as in circulating hematopoietic cells. Angiotensin II was found to interact with the AT1 receptor to stimulate erythroid differentiation and increase hematopoietic progenitor cell proliferation. Moreover, stimulation of AT1/AT2 receptors was found to exert an stimulatory/inhibitory action on JAK-STAT pathway, which is directly linked to physiologic functions of erythropoietin, thrombopoietin and other cytokines. Finally, ACE was implicated in enhancing the recruitment of primitive stem cells into the S-phase by degrading AcSDKP. We have tested the AGTRAP function in hematopoietic cells. For that purpose we overexpressed AGTRAP protein in the K562 cells and in K562 cells driven by Mpl signaling. AGTRAP overexpression stimulated the growth of plain K562 cells decreasing the cells division time from 16 to 12 hours. Importantly, it synergized with Mpl signaling to promote the proliferation rate of Mpl dependent K562 cells (expressing the dimerizable construct of Mpl activated with AP20187 while the transforming Bcr-Abl protein was inactivated with Imatinib) decreasing the cells division time from 13 to 9.6 hours. The survival of Mpl dependent K562 cells over-expressing AGTRAP was still dependent on Mpl signaling and on Jak2 function as tested by withdrawal of AG20187 and by treatment with Jak2 inhibitor AG490. Overexpression of AGTRAP protein over-sensitized the UT7/TPO cells to treatment with recombinant human megakaryocyte growth and development factor (rhMGDF), increasing growth rate of the cells at low rhMGDF concentrations. The cells proliferation rate was increased by 22.5% and 39.3% at 1/100 and 1/1000 of optimal concentrations of rhMGDF respectively. There was no growth advantage from AGTRAP expression at optimal concentration of rhMGDF, however in the presence of AGTRAP the UT7/TPO cells were able to proliferate in the absence of rhMGDF altogether. Surprisingly, the overexpression of AGTRAP negatively regulated the growth of HEL cells, known to carry a homozygous transforming mutation of Jak2V617F. In conclusion our data implicate the RAS member AGTRAP in hematopoietic cell proliferation and survival and point to its synergistic effect with the proliferation promoting function of Mpl. Disclosures: No relevant conflicts of interest to declare.

Local bone marrow renin–angiotensin system in primitive, definitive and neoplastic haematopoiesis

“…The RAS member AGTRAP (AngII-receptor-associated protein) is active in haematopoietic cell proliferation and survival and points to its synergistic effect with the proliferation promoting function of the thrombopoietin receptor Mpl [9]. The Mpl protooncogene plays a basic role in megakaryocytic development and platelet production, as well as performing a substantial function in HSC homoeostasis and self-renewal [68].…”

“…The results on angiotensin peptides in thrombopoiesis is very limited. The only study is by Kwiatkowski et al [9], who performed retroviral insertional mutagenesis using an MSCV (murine stem cell virus)-based vector coding for a drug-dependent dimerizable fusion protein that contains the cytoplasmic domain of Mpl. They used the vector construct to transduce the human leukaemia cell line K562, which also includes all of the RAS elements [45].…”

“…Locally active BM RAS affects critical steps of physiological and pathological blood cell production via autocrine, paracrine and intracrine pathways [4,5]. Haematopoietic niche [6], myelopoiesis [7], erythropoiesis [8], thrombopoiesis [9] and the development of other cellular lineages [6,[10][11][12] are regulated by the biological actions of local BM RAS peptides. The local RAS [4,5] Abbreviations: ACE, angiotensin-converting enzyme; ACEI, ACE inhibitor; AcSDKP , N-acetyl-Ser-Asp-Lys-Pro; AGM, aorta-gonad mesonephros; AML, acute myeloid leukaemia; Ang-(1-7), angiotensin-(1-7); AngI etc., angiotensin I etc.…”

“…The major RAS effector mediator AngII (angiotensin II) exerts its haematopoietic effects by activating angiotensin receptors, primarily AT 1 Rs and AT 2 Rs, within the BM micro-environment [4,5]. ACE (CD143) has been implicated in enhancing the recruitment of primitive stem cells into S-phase by degrading AcSDKP [9,[14][15][16][17]. Moreover, stimulation of AT 1 Rs/AT 2 Rs by AngII, the principle effector molecule of the RAS, exerts stimulatory/inhibitory action on the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway, which is directly linked to the physiological functions of erythropoietin, thrombopoietin and other haematopoietic cytokines during normal haematopoiesis and in myeloproliferative neoplasms [9,[18][19][20].…”

“…ACE (CD143) has been implicated in enhancing the recruitment of primitive stem cells into S-phase by degrading AcSDKP [9,[14][15][16][17]. Moreover, stimulation of AT 1 Rs/AT 2 Rs by AngII, the principle effector molecule of the RAS, exerts stimulatory/inhibitory action on the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway, which is directly linked to the physiological functions of erythropoietin, thrombopoietin and other haematopoietic cytokines during normal haematopoiesis and in myeloproliferative neoplasms [9,[18][19][20]. The local RAS is effective even at the stage of primitive embryonic haematopoiesis [21][22][23][24].…”