GAS2is Up-regulated In CML Cells And Required For Their Growth

Objective：This study aims to delineate the role of growth-arrest specific2(GAS2) inCML.Methods:（1）The nucleated cells of CML patients and healthy donors were obtainedwith gradient centrifuge using Ficoll, and then these cells were purified withimmnomagnetic method to enrich CD34+cells. lin-CD34+and lin-CD34+CD38-cellswere purified with FACS. The transcript and protein expression were detected withQ-RT-PCR and immunoflurorescence.（2） Lentiviral vectors were utilized to delivershRNAs against GAS2to K562, MEG-01and CD34+cells from CML patients, then thecalpain activity, growth and the colony-forming cell (CFC) abilities of GAS2silenced andcontrol cells were compared.（3）A truncated mutant of GAS2(GAS2△171-313, thedominant negative form of GAS2, GAS2DN) was amplified with PCR and then deliveredwith a lentiviral vector to K562, MEG-01and CD34+cells from CML patients. Thecalpain activity, growth and the colony-forming cell (CFC) abilities of GAS2DN expressedand control cells were compared.（4）The sensitivity of GAS2silenced cells and GAS2DNexpressed cells in the treatment of Imatinib mesylate (IM） were measured and comparedto their control cells.（5）The tumorigenic capacities of GAS2DN expressed and controlMEG-01cells were compared, when they were injected subcantenously to nude mice.（6）To delineated the role of calpain activation in the inhibited growth of cells upon GAS2DN,the interaction of GAS2and calpain was verified with co-immunoprecipitate, and calpainwas silenced with a lentiviral vector delivered shRNA against Calpain.（7） Thetransactivation activity of β-catenin was analyzed with TOPFlash/FOPFlash reporterplasmids in GAS2silenced and GAS2DN expressed cells compared to their control cells.（8）The transcriptome data were generated and compared with other public accessibledatabase to obtain molecular insights of how GAS2DN inhibits the growth of CML cells.（9）Some candidate differentially expressed genes upon GAS2DN were validated with Q-RT-PCR and western blot, the function of one candidate gene in the growth of CMLcells was studied with the silence of its expression by RNAiResults: The transcript of GAS2was significantly higher in leukemic cells comparedwith their normal control cells. Similar data were obtained with the comparison ofleukemic and normal cells using immunofluorescence. We validated two independentshRNA sequences, which could inhibit the expression of GAS2effectively and lead to theactivation of Calpain. GAS2silenced K562, MEG-01and CD34+cells grew slower thantheir control cells, and produced less CFC than control cells as well. On the other hand, wegenerated a lentiviral vector to deliver GAS2DN (GAS2△171-313, a dominant negativeform of GAS2) to CML cells, which activated Calpain and led to the inhibited growth andCFC production. Interestingly, the inhibition of leukemic CD34+cells were significantlystronger than that of CD34+cells form the healthy donors. GAS2DN was capable tosuppress the tumorigenic abilities of MEG-01cells (control group:10/12versus GAS2DNgroup：3/10) when they were injected subcautenously to nude mice. In addition, the growthrate of the tumors form GAS2DN group was significantly slower than those from thecontrol group. We also found that targeting GAS2enhanced the sensitivity of K562cells inthe treatment of IM.GAS2and Calpain2were confirmed to interact in CML cells. The inhibition ofCalpain2with RNAi successfully rescued the suppressed CML cell growth upon GAS2DN.In this study, we found that targeting GAS2did not affect the expression and thelocalization of β-catenin. Indeed, we could not detect the transaction activity of β-cateninin both K562and MEG-01cells either.In order to obtain molecular insights of how targeting GAS2inhibits the growth ofCML cells, we generated transcirptome data to compare GAS2DN expressed and controlMEG-01cells. Through the meta-comparison with other public accessible database, wevalidated that the expression of HNRPDL, PTK7and UCHL5were suppressed uponGAS2DN. Interestingly, these3genes were all up-regulated in both nucleated cells andCD34+cells from CML compared to those from healthy donors. Lastly, we found silenceof HNRPDL was capable to inhibit the growth of K562cells and sensitize these cells in thetreatment of IM.Conclusion: GAS2(Growth-arrest specific2) has significantly higher expressedtranscript and protein in both nucleated cells and CD34+cells from chronic myeloid leukemia patients in comparison to those from healthy donors.Targeting GAS2(RNAi and the expression of GAS2DN) is capable to activateCalpain, lead to the inhibition of CML cells growth and CFC production and enhance theIM sensitivity of CML cells. In addition, GAS2DN inhibits the tumorigenic activity ofMEG-01cells in nude mice. Interestingly, GAS2DN has stronger inhibitory effect onCD34+cells from CML patients than those from healthy donors. In the present study, wealso show that the inhibiton of CML cells by targeting GAS2is partially Calpain dependent,but not the degradation of β-catenin, possibly through the down-regulation of a set of genesincluding HNRPDL.Taken together, our data show that GAS2is up-regulated in CML cells and plays anovel regulatory role in the growth of these cells, which possibly serves as a noveltherapeutic target to improve the treatment of CML.