The Role And Underlying Mechanism Of Transcriptional Regulatory Molecules RBP2 And YAP1 In Adult Leukemia

BackgroundLeukemia is a life-threatening neoplasm of hematopoietic stem cells. In the bone marrow and other hematopoietic tissues, leukemia cells lose the ability to differentiate into mature cells and lead to the expansion of immature cells in the bone marrow. Leukemia can be divided into acute and chronic leukemia.Acute lymphoblastic leukemia (ALL) is a clonal malignancy of lymphocyte precursors (B-cell or T-cell), characterized by uncontrolled growth and leukemic expansion of immature lymphoblastic progenitor cells.This disease prevails in both children and adults, with peak incidence between the ages of 2 to 5 years. In recent years, multiagent treatment regimens can cure more than 80% of children diagnosed with ALL. In contrast, the prognosis for adults ALL is significantly worse and it is easy to relapse after complete remission. ALL is a heterogeneous disease with multiple, prognostically relevant genetic aberrations. The mechanism of ALL is still not clear. For many years, leukemia was thought to stem primarily from genetic alterations;however, epigenetic alterations have also been recognized as a major driving force in the development of ALL.Retinoblastoma binding protein-2 (RBP2), also called KDM5A or JARID1A, is one of the members of j arid family which accounts for histone demethylase (HDM) activity. RBP2 specifically target tri- and di-methylatedlysine 4 of histone H3 (H3-K4) for demethylation. Recently RBP2 was identified as one chromatin-modifying enzyme that participates in the carcinogenesis and progression of human cancers.RBP2 can affect cell proliferation, senescence and differentiation and promotes the development and progression of gastric cancer, liver cancer, lung cancer and other solid tumor.However, our previous study showed that RBP2 played the role of tumor suppressor in CML. Thus, RBP2 has dual role as an oncogene or tumor suppressor. Whether RBP2 plays a key role in ALL remains undetermined.Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by splenomegaly, anemia and granulocytosis. t (9; 22) (q34; qll) chromosome translocation lead to the BCR-ABL fusion gene which encodes BCR-ABL fusion protein. CML is triphasic:chronic phase (CP), accelerated phase (AP) and blastic phase (BP). Tyro sine kinase inhibitor (TKI) is the first-line treatment of CML, which includes the first generation of TKI (imatinib), the second generation of TKI (dasatinib and nilotinib) and the third generation of TKI (ponatinib). However, there are still 15%-20% patients are not sensitive to tyrosine kinase inhibitor, and 10%-15% patients undergo blast crisis at diagnose. Once CML transforms into an acute-type leukemia undergoing a’blast crisis’, the disease becomes highly aggressive and incurable. However, the mechanisms of the CML blast transformation are still unclear. BCR-ABL is the main driving force for both CML-CP and CML-BP. However, in recent years, many studies showed that BCR-ABL independent mechanisms also played important role in CML progression, including inactivation of tumor suppressor genes and activation of oncogenes.Yes-associated protein 1 (YAP1) as an important regulator of Hippo pathway, plays important role in controlling organ size and was involved in carcinogenesis. YAP1 is a transcriptional coactivator that regulates target genes by interacting with transcriptional regulators. YAP1 plays oncogenic role mainly through interacting with transcriptional activator and TEAD family. However, YAP1 plays a dual role as oncogene and tumor suppressor in human tumors. YAP1 promotes the development and progression of gastric cancer, lung cancer and liver cancer, etc. Recent researches showed that YAP1 is downregulated in promyelocytic leukemia (PML) and multiple myeloma(MM) through interacting with P73 and triggering DNA damage. YAP1 also played tumor suppressor role in colon cancer and breast cancer. Therefore, the role of YAP1 involved in oncogenesis depends on the tissue specificity. Whether YAP1 plays a key role in CML blast transformation remains undetermined.AIM1. We explored the role and mechanism of RBP2 in adult acute lymphoblasticleukemia.2. We explored the role of YAP1 in blast crisis of chronic myeloid leukemia. Methods and Results1. RBP2 promotes adult ALL by upregulating BCL2.(1) RBP2 is overexpressed in ALL:We collected bone marrow samples from 42 newly diagnosed (de novo ALL),36 complete remission (ALL-CR),9 relapsed (ALL-relapse) and 33 iron deficiency anemia (con) adult patients. RBP2 was examined by qRT-PCR, Immunofluorescence (IF) and Western Blot, the results showed that RBP2 was uniformly expressed at high levels in de novo ALL and reduced to normal levels upon complete remission. Once relapsed, RBP2 re-expressed. Then, we analyzed 4 matched diagnostic-remission bone marrow samples to determine the role of RBP2 in ALL progression. qRT-PCR of these samples revealed that RBP2 downregulated significantly by the time that the patients underwent complete remission.(2) RBP2 knockdown promoted cell apoptosis and inhibited cell proliferation:We transfected Jurkat cells with specific RBP2-siRNA and lenti-RBP2-shRNA virus. As expected, RBP2 knockdown resulted in a significant increase of apoptosis rate in Jurkat cells and decrease of cell proliferation and colony-formation ability.(3) BCL2 is a novel target of RBP2:In both Jurkat cells and ALL patient primary cells, qRT-PCR and Western Blot showed that RBP2 knockdown reduced the expression of BCL2. To further explore the correlation between RBP2 and BCL2, we explored their mRNA expressions in ALL samples. As expected, their mRNA levels showed positive correlation. To validate if BCL2 is a direct target of RBP2, we divided the BCL2 promoter which contains RBP2 binding sites into a, b, c regions, CHIP assay showedRBP2 bound to the b region but no binding to a or c region. BCL2 promoter luciferase reporter plasmid system showed RBP2 knockdown inhibited the transcriptional activity of BCL2 promoter.(4) BCL2 mediates RBP2-promoted proliferation and -inhibited apoptosis and RBP2 inhibition sensitize Jurkat cells to ABT-199:Flow cytometry and EdU showed that BCL2 expression partially reversed the role of RBP2 knockdown and RBP2 knockdown increased the sensitivity of Jurkat cells to BCL2 inbihitor ABT-199.2. YAP1 is a tumor suppressor in blast crisis of CML.(1) YAP1 is downregulated in blast crisis of CML:First, we analysed the gene expression assays of CML patients in GEO DataSets and found YAP1 is decreased in blast crisis of CML patients compared with chronic phase.We investigated the expression of YAP1 in bone marrow samples from38 CML-CP patients and 21 CML-BP patients. As expected, IF and qRT-PCR showed that YAP1 expresssion reduced in BP patients compared to CP patients.(2) YAP1 promoted cell apoptosis and differentiation:We transfected CML cells with YAP1 expression plasmid and lenti-YAP1 virus. As expected, YAP1 overexpression resulted in a significant increase of apoptosis rate and decrease of cell proliferation and led to the upregulation of P73 targetgenes (P21 and Bax). After the K562 and MEG-01 cells undergoing megakaryocytic differentiation by PMA, qRT-PCR and Western Blot showed that YAP1 upregulated at both mRNA and protein level. Also, qRT-PCR and flow cytometry showed that the megakaryocytic marker CD61 increased after YAP 1 overexpression.(3) PDCD4 is a target gene of YAP 1:qRT-PCR and Western Blot showed that YAP1 did not regulate the expression of BCR-ABL, but upregulated PDCD4.(4) YAP1 is an epigenetic regulated tumor suppressor:qRT-PCR and Westeren Blot showed that both DNA methylase inhibitor (decitabine) and histone deacetylase (HDAC) inhibitor (chidabine) could upregulate the expression of YAP1 but not through the Hippo pathway.ConclusionsRBP2 and YAP1 are both dual transcriptional regulators, our results showed that: RBP2 promotes adult ALL by upregulating BCL2; YAP1 decreased during the blast transformation of CML and promotes cell apoptosis and megakaryocytic differentiation. These results indicate that RBP2 and YAP1 are potential markers for leukemia diagnosis, treatment and monitoring the leukemia progression.