ALBERT

Description: Runx1/AML1 is a key regulator of hematopoiesis and leukemic transformation, as RUNX1(−/−) mice do not develop definitive hematopoietic stem cells, and sever alleukemic oncogenes, e.g. AML1-ETO, CBFβ-SMMHC, or TEL-AML1, inhibit Runx1activities. We have investigated regulation of cell cycle progression by Runx1. Runx1stimulates G1 to S cell cycle progression in hematopoietic cell lines and in transduced myeloid progenitors, and inhibition of Runx1 by CBFβ-SMMHC or AML1-ETO slows G1 progression. Runx1 induces cdk4 and cyclin D3 transcription, and exogenous cdk4, cyclin D2, or c-Myc overcomes inhibition of G1 progression by CBF oncoproteins. In addition to regulating cell cycle progression, Runx1 protein levels are themselves increased as hematopoietic cells progress from G1 to S to G2/M, though mRNA levels remain constant. Runx1 contains three consensus cdk sites, (S/T)PX(R/K), S48, S303, and S424, and using phospho-specific antisera we find that each of these is modified in hematopoietic cells. Mutation of these serines to aspartic acid, mimicking phosphorylation, increases trans-activation of a reporter containing four CBF sites or the TCRβ promoter, whereas mutation to alanine reduces trans-activation. p300 interacts similarly with Runx1(tripleA) and Runx1(tripleD). We have now evaluated interaction of HDACs1–8 with these variants and Runx1 and find that both HDAC1 and HDAC3 have reduced affinity for RUNX1(tripleD), as assessed by co-immunoprecipitation from transiently transfected 293T cells. Evaluation of single serine residue mutants (S48D, S303D, and S424D) demonstrates reduced affinity of HDAC1 or HDAC3 specifically for the Runx1(S424D) mutant, consistent with previous mapping of the Runx1:HDAC1 and Runx1:HDAC3 interactions to this region of Runx1. Thus, cdk phosphorylation of Runx1 S424 reduces affinity for HDAC1 and HDAC3, increasing Runx1 trans-activation potency. Regulation of Runx1 activity by cdks may control key developmental processes, including expansion of definitive HSC during development and regulation of the balance between adult HSC quiescence and proliferation.

Description: C/EBPα mediates myeloid differentiation, and its reduced activity is central to myeloid transformation (Friedman Int. J. Hematol. 2015). The murine Cebpa gene contains a 450 bp, +37 kb element that acquires the enhancer-specific H3K4me1 and H3K27Ac histone marks as LT-HSC progress to GMP and directs hCD4 transgene expression to GMP and myeloid CFUs (Guo et al. Blood 2012; Guo et al. J. Leuk. Biol. 2014). Moreover, CRISPR/Cas9 mediated, biallelic replacement of the enhancer with a variant harboring mutations in its seven Ets sites reduces Cebpa RNA 〉10-fold in 32Dcl3 cells (Cooper et al. PLoS One 2015), and germline or Cre-mediated enhancer deletion leads to marked reduction in Cebpa RNA in marrow LSK, CMP, and GMP, with 3-fold reduction in GMP, CFU-G, and neutrophils and indefinite myeloid colony replating in IL-3, a preleukemic phenotype (Guo et al. PLoS One 2016; Avellino et al. Blood 2016). The enhancer contains four conserved RUNX1 cis elements that bind RUNX1 in gel shift or ChIP assays, Runx1 gene deletion reduces Cebpa RNA 5-fold in Lin- and 2-fold in GMP marrow cells, Runx1 cis element mutation reduces luciferase reporter activity 6-fold in 32Dcl3 myeloid cells (Guo et al. Blood 2012), and in AMLs with t(8;21) RUNX1-ETO interacts with the human CEBPA locus at the homologous +42 kb enhancer (Ptasinska et al. Leukemia 2012). Mutation of the CEBPA enhancer has not been seen in human AML cases, perhaps reflecting preference for upstream pathway alteration to suppress both alleles and additional genes. As a further example of a leukemic alteration affecting enhancer activity, elevated EVI1 is a high-risk feature in AML, and EVI1 binds and represses the +37 kb Cebpa enhancer (Wilson et al. J. Biol. Chem. 2016). We have characterized additional pathways whose modulation in AML may reduce CEBPA enhancer activity. Deletion of the Pu.1 -14 kb enhancer leads to AML in mice, and PU.1 binds the Cebpa enhancer in ChIP and gel shift; we now find that mutation of the one Cebpa enhancer Ets site that binds PU.1 reduces reporter activity 4-fold in 32Dcl3 cells, providing the first functional evidence that PU.1 regulates the enhancer. ~10% of human AMLs harbor mutant C/EBPα proteins, CEBPα binds the enhancer in ChIP and gel shift, and mutation of the two enhancer C/EBP elements reduces reporter activity; we have now used CRISPR/Cas9 to generate a 32Dcl3 cell line with the two enhancer C/EBPsites mutated in one allele and find 60% reduction in Cebpa RNA expression. FLT3/ITD is a constitutively activated mutant form of the receptor tyrosine kinase found in ~30% of AML cases and confers high risk. While FLT3/ITD predominantly provides proliferative signals, it may also contribute to impaired differentiation, as FLT3/ITD inhibition in AML cases leads to a neutrophilic differentiation syndrome. We previously demonstrated that FLT3/ITD reduces Cebpa RNA in 32Dcl3 cells and patient leukemic blasts and that the first generation FLT3/ITD inhibitor lestaurtinib increases Cebpa; we now find that the effect on Cebpa RNA is also reversed in 32Dcl3 cells by the next generation inhibitor crenolanib, that FLT3/ITD reduces Cebpa enhancer H3K4me1 and H3K27Ac histone marks several-fold, and that both of these activating marks are also restored by crenolanib. FLT3/ITD signaling leads to C/EBPα serine phosphorylation to reduce its activity, which may account in part for the inhibitory effect of FLT3/ITD on Cebpa enhancer activity. Finally, NUP98-HOX fusion proteins contribute to a small percent of AML cases. Vav-NUP98/HOXD13 (NHD13) mice develop MDS and can progress to AML; we now find that Cebpa RNA is reduced 5-fold in Vav-NHD13 CMP and LSK, with 5-fold increased HoxA9 RNA, that Vav-NHD13::Cebpa Enh-hCD4 compound heterozygous mice have reduced hCD4 expression in GMP and CMP, implicating a direct effect on enhancer activity, and that expression of NHD13 in 32Dcl3 subclones prevents G-CSF induction of Cebpa, MPO, and morphologic differentiation. These 32Dcl3 lines retain levels of Gcsfr equal to empty vector-transduced cells in IL-3 and manifest increased HoxA9 in response to G-CSF. Moreover, HoxA9 binds the Cebpa enhancer in ChIP using marrow-derived HoxA9/Meis1 myeloid lines. Potentially, NHD13 induces HoxA9, which then binds the Cebpa enhancer directly or via other factors to repress transcription. Identifying mechanisms that repress CEBPA hematopoietic enhancer activity in AML may identify approaches to induce differentiation. Disclosures No relevant conflicts of interest to declare.

Description: The C/EBPα transcription factor is a key mediator of normal myeloid differentiation, and its reduced expression or activity is central to myeloid transformation, as recently reviewed (Friedman, Int. J. Hematol. 2015). The murine Cebpa gene contains a conserved, 450 bp, +37 kb regulatory element that displays the enhancer specific H3K4me1 and the activating H3K27Ac histone modifications at increasing levels as LT-HSC progress to ST-HSC, CMP, and GMP, increases promoter activity 6-fold when introduced into 32Dcl3 myeloid cells linked to a luciferase reporter, directs murine hCD4 transgene expression preferentially to GMP, myeloid CFUs, and functional LT-HSC, and is bound and activated by RUNX1, PU.1, SCL, GATA-2, C/EBPα, and c-Myb (Guo et al, Blood 2012; Guo et al, J. Leuk. Biol., 2014; Cooper et al, PLoS One 2015). In addition, CRISPR/Cas9 mediated enhancer mutation markedly reduces Cebpa transcription in 32Dcl3 cells (Cooper et al, 2015), and in AML samples harboring t(8;21) the RUNX1-ETO oncoprotein interacts with the human CEBPA locus specifically at the homologous +42 kb enhancer. We have now characterized mice harboring floxed Cebpa +37 kb enhancer alleles. Utilizing a B6 BAC encompassing the Cebpa locus, we assembled a targeting vector containing a 5.5 kb 5' homology arm, the +37 kb enhancer flanked by lox P sites, a PGK-Neo cassette flanked by frt sites, and a 0.9 kb 3' homology arm. After introduction into a B6 mESC line, several G418-resistant subclones were isolated, and homologous, heterozygous targeting was confirmed by 3' PCR and 5' and 3' Southern blotting. Two ESC lines were utilized to develop germ line chimeras, and upon breeding with B6 albino mice, one yielded several offspring with germ line transmission of the targeted allele, which were then bred to homozygosity. In an initial set of experiments, marrow from wild-type (WT) and Enh(f/f) mice exposed to 5-FU was transduced with pBabePuro or pBabePuro-Cre followed by puromycin selection and in vitro analysis. Cebpa mRNA was reduced 10-fold in the lineage-negative subset upon Enh(f/f) marrow transduction with Cre. CFU-G were reduced 8-fold, whereas CFU-M and CFU-GM were unaffected, and enhancer deletion enabled serial replating of myeloid CFUs for 〉16 generations, though these remained dependent on IL-3. Morphologically, enhancer deletion led to depletion of mature granulocytes and accumulation of CD11b- Gr-1- blasts after four days of culture in IL-3, IL-6, and SCF. We next generated Enh(f/f);Mx1-Cre mice and subjected these and Enh(f/f) littermates to pIpC injections, followed by analysis four weeks after the last injection. Cebpa mRNA was reduced 12-fold in GMP, 13-fold in CMP, and 〉20-fold in the LSK marrow subset. Peripheral blood and marrow neutrophils were reduced 3- to 8-fold, whereas monocytes, RBCs, platelets, and B or T lymphocytes were unaffected. GMP as a percentage of Lin- Sca-1- c-kit+ progenitors were reduced 3-fold, CMP were increased 1.5-fold, and MEP and CLP were unaffected. CFU-G were reduced 3-fold, CFU-M were increased 2-fold, and BFU-E were increased 4-fold. LSK as a percentage of Lin- cells were increased 3-fold, and MPP and ST-HSC as a percentage of LSK were unaffected, whereas LSK/SLAM were reduced 10-fold, and enumeration of functional LT-HSC by competitive transplantation is in progress. We also generated Enh(f/f);Vav-Cre mice; these demonstrated similarly impaired granulopoiesis, but were obtained at only 12% of the Mendelian ratio, suggestive of embryonic lethality as early death was not observed. In addition, we used FLPo-Cre to delete the Neo cassette; homozygous deletion did not alter myeloid CFU numbers or FACS-defined progenitors. Finally, we utilized CMV-Cre to develop germ line enhancer-deleted mice. As with Vav-Cre, homozygous offspring were obtained far below the predicted numbers. Nevertheless, these expressed Cebpa mRNA at levels equivalent to WT controls in liver, lung, adipose, kidney, small intestine, skeletal muscle, and cardiac tissues, and these organs displayed normal cellular architecture upon hematoxylin-eosin staining. In summary, conditional deletion of the +37 kb Cebpa enhancer in adult mice demonstrates that it acts as a critical, hematopoietic specific enhancer of Cebpa gene expression and that marked Cebpa mRNA down-modulation upon enhancer deletion impairs granulopoiesis and enables serial, cytokine-dependent myeloid CFU replating, a pre-leukemic phenotype. Disclosures No relevant conflicts of interest to declare.

Description: Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P〉0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P

Description: Deletion of Runx1 in adult mice produces a myeloproliferative phenotype. We now find that Runx1 gene deletion increases marrow monocyte while reducing granulocyte progenitors and that exogenous RUNX1 rescues granulopoiesis. Deletion of Runx1 reduces Cebpa mRNA in lineage-negative marrow cells and in granulocyte-monocyte progenitors or common myeloid progenitors. Pu.1 mRNA is also decreased, but to a lesser extent. We also transduced marrow with dominant-inhibitory RUNX1a. As with Runx1 gene deletion, RUNX1a expands lineage−Sca-1+c-kit+ and myeloid cells, increased monocyte CFUs relative to granulocyte CFUs, and reduced Cebpa mRNA. Runx1 binds a conserved site in the Cebpa promoter and binds 4 sites in a conserved 450-bp region located at +37 kb; mutation of the enhancer sites reduces activity 6-fold in 32Dcl3 myeloid cells. Endogenous Runx1 binds the promoter and putative +37 kb enhancer as assessed by ChIP, and RUNX1-ER rapidly induces Cebpa mRNA in these cells, even in cycloheximide, consistent with direct gene regulation. The +37 kb region contains strong H3K4me1 histone modification and p300-binding, as often seen with enhancers. Finally, exogenous C/EBPα increases granulocyte relative to monocyte progenitors in Runx1-deleted marrow cells. Diminished CEBPA transcription and consequent impairment of myeloid differentiation may contribute to leukemic transformation in acute myeloid leukemia cases associated with decreased RUNX1 activity.

Description: Objective Previous researches confirmed that multidrugresistance(MDR) plays an important role in the failure of chemotherapy on malignant tumors. This study was to investigate the molecular biological mechanisms of cyclosporine A(CsA), tetrandrine(Tet) and their combination on multidrug resistance cell line K562/A02. Methods K562/A02 cells were treated with cyclosporine A and (or) tetrandrine. The intracellular DNR concentration and the expression of P-glyco-protein (P-gp) were observed by flow cytometry (FCM) assay. The mRNA expression of mdr-1 was measured by fluorescent semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR). Resulds CsA and Tet (alone or combination) elevated the intracellular DNR concentration in K562/A02 cells(the fluorescence intensity of intracellelar DNR in K562/A02 cells was 60%,65% and 98% respectively of that in K562 cells); The fluorescence intensity of P-gp in K562 and K562/A02 cells was 0.5% and 97.97%. The P-gp expression was down after treated with CsA,Tet and both(75.32%,76.86% and 48.61%); mdr1 mRNA was also down regulated, and the effect of their combination was greater. Conclusion Multidrug resistance (MDR) can be partially reversed by CsA or Tet, the combination of both drugs shows a great synergistic reversal effect.

Description: Object: To study the variation of p21, P-gp expression in reversion of multidrug resistance of K562/A02 leukemic line with different concentration of tetrandrine(TET), to provide new theoretic evidence for the clinical application of TET. Methods: The IC50 (the concentration causing 50% inhibition of cell growth) of DNR was analyzed by MTT method; The Expressions of p21 were assayed by westernblot; The Expressions of P-gp were assayed by FCM; The expressions of mdr1 were assayed by RT-PCR; The variation of p21 was accentuation with the accrescence concentration of TET(P

Description: Objective: To establish the xenograft leukemia model with stable multiple drug resistance in nude mice; to investigate the reversal effect of 5-Bromotetrandrine and Magnetic nanoparticle of Fe3O4 combined with DNR in vivo and to search for the possible reversal mechanisms. Methods: K562 and K562/A02 cells were respectively inoculated subcutaneously into back of athymic nude mice (1×107 cells/each) to establish the xenograft models. The tumor formation was evaluated by animal ultrasonic inspection. Tumors-bearing nude mice were assigned randomly to five groups which were treated with NS (A group); DNR 1mg/kg (B group); nanoparticle of Fe3O4 combined with DNR 0.63mg/kg(C group): 5-BrTet 2.5mg/kg combined with DNR(D group); 5-Bromotetrandrine 2.5mg/kg and Magnetic nanoparticle of Fe3O4 combined with DNR 0.63mg/kg(E group) respectively. The incidence of tumor formation, growth characteristics, weight and volume of tumor were observed. The histopathologic examination of tumors and organs were detected. For resistant tumors, the protein levels of P-glycoprotein (P-gp) were detected by Western blot. Results: The tumor incidence was 100% in the nude mice inoculated with either K562 or K562/A02 cells. In 6 to 9 days,the tumors reached a volume of more than 1 00 mm3. In vivo, MTT assay showed K562/A02 tumor maintained the drug resistance. For K562 cells xenograft tumors, there were no apparent differences in tumor suppression effect between the B AC AD AE group. For K562/A02 cells xenograft tumors, 5-BrTet and Magnetic nanoparticle of Fe3O4 combined with DNR significantly suppressed growth of tumor: the inhibition rate was 62.76% while DNR alone be used, the inhibition rate was 3.68%. Pathologic examination of resistant tumors showed the tumors necrosis obviously in E group. Application of 5-BrTet and Magnetic nanoparticle of Fe3O4 inhibited the overexpression of P-gp. Conclusion: The xenograft leukemia nude mice model was maintain the multiple drug resistance. 5-Bromotetrandrine and Magnetic nanoparticle of Fe3O4 combined with DNR had a significant tumor-suppressing effect on MDR leukemia cells xenograft model.

Description: Objective To evaluate the MDR reversal activity of magnetic nanoparticle Fe3O4 (Nano-Fe3O4) and 5-bromotetrandrine (BrTet) on multidrug resistance cell line K562/A02 solitarily or symphysially, and to investigate the reversal mechanism of this coopration. Methods The proliferation of K562 and K562/A02 cells which were cultured with daunomycin (DNR) alone or in combination with Nano-Fe3O4, BrTet or both for 48h were evaluated by MTT assay. DNR accumulation and P-gp of K562 and K562/A02 were analyzed by fluorospectrophotometry. Results The IC50 of DNR for K562 and K562/A02 cells were 2.74±0.19μM and 32.33±8.40μM, respectively; but the sensitivity of K562/A02 cells to DNR was partially restored after culturing with Nano-Fe3O4, BrTet solitarily and symphysially (the IC50 were 7.04±0.85μM, 4.25±2.16μM, 1.80±0.30μM; the FR were 4.32, 7.61, 17.96, respectively), but had no effects on K562 cell lines. The differences had statistical significance. Flow cytometry assay showed that Nano-Fe3O4 and BrTet increased the DNR accumulation in K562/A02 cells, especially in the group of synergia of these two agents. The mean fluorescence intensity of endocellular DNR increased from 2614 pretreated with DNR only to 4783 incubated with these two reversal reagents, while the values were 3364, 4077 for incubating with Nano-Fe3O4 and BrTet respectively. P-gp were down regulated through pretreating with Nano-Fe3O4, BrTet alone and symphysially in K562/A02 cells by fluorospectrophotometry assay. Conclusion Nano-Fe3O4 and BrTet synergisticly showed significant MDR reversal activity in vitro. The reversal activity may be related to the inhibition of P-gp overexpression and the increasing of the anticancer drug accumulation intracelluarly.

Description: Objective: To observe the apoptosis of K562/A02 cells induced by application of Fe3O4-Magnetic Nanoparticle (Fe3O4-MNPs) loaded with Daunorubicin(DNR) and 5-Bromotetrandrine (BrTet) and to explore its possible molecular mechanism. Methods: We detected the apoptosis of K562/A02 cells after treatment with Fe3O4-MNPs and BrTet with DNR alone or their combination by flow cytometry(FCM),Wright staining and DNA ladder. The expression of Bax and Bcl-2 protein was measured by Western blot. Results: The result showed apoptotic characteristics after treated with this composite for 48 hours. Flow cytometry assay showed the percentage of apoptotic cells which were treated with Fe3O4-MNPs and BrTet with DNR alone or their combination for 48 hours increased from (7.9%±0.35)% to (15.5%±0.87)%, (19.7%±1.04)% and(40.8%±1.74)%, (P