Hematological Malignant Cell Lines Research Articles

Lncrna RP1-68D18.2 Promotes K562 Cell Line Resistance to Imatinib through CD44 and Its Downstream Signal Molecules

As the first-generation tyrosine kinase inhibitor specifically targeting the BCR-ABL oncoprotein, imatinib has been successfully used in the therapy for chronic myelogenous leukemia (CML) with a significant response and good compliance. However, imatinib resistance becomes the main reason for failure of the treatment of CML. In addition to the common BCR-ABL dependence of imatinib resistance, e.g. BCR-ABL kinase domain mutations and BCR-ABL gene amplification, BCR-ABL independence also plays important roles, which involves alteration of dynamics of drug import and efflux, persistent or compensatory activation of signaling pathways etc. Recently, several studies have reported the correlation of lncRNA with resistance of chemotherapeutic drugs in many cancers. Here, we report, for the first time, discernible overexpression of lncRNARP1-68D18.2 and CD44 mRNA in the imatinib resistant chronic myelogenous leukemia cell line K562R, using lncRNA and mRNA microarray, and further verify it using qPCR. Knockdown of RP1-68D18.2 in K562R cell lines increased their sensitivity to imatinib, and simultaneously caused a decrease in CD44 and c-Myc protein levels. Correspondingly, overexpression of RP1-68D18.2 in K562, a sensitive cell line, led to a slightly decrease in imatinib sensitivity and partially reverse downregulation of c-Myc, p-ERK and p-MEK caused by imatinib treatment. Analysis of correlations revealed positive correlation between RP1-68D18.2 and CD44 mRNA expression in the primary CML blasts and hematological malignant cell lines. We conclude that overexpression of RP1-68D18.2 could promote imatinib resistance in the K562R cell line through the regulation of CD44 and its downstream signal molecules. DisclosuresNo relevant conflicts of interest to declare.

In Vitro Approach for the Identification of Exceptional Responders in Acute Myeloid Leukemia

Each case of Acute Myeloid Leukemia (AML) represents a unique ecosystem. Despite recent advances in our understanding of the genetic landscape of AML, this information remains insufficient to accurately match patients with targeted therapies. While pediatric and adult AML share phenotypic similarities, pediatric AML represents a genetically distinct disease from adult AML, and will benefit from independent genomic studies and novel therapeutic strategies. Real-time ex-vivo functional screening can identify mechanisms underpinning drug response and diversity between tumors, aiding in patient stratification. We established an in vitro drug screening system that incorporates cytokine signaling to better model inflammation, stem cell function, and niche derived support of leukemic blasts. This approach provided insight into variability between patients who currently would be placed on the same therapeutic regimen.Samples were acquired from 12 pediatric AML patients, after informed consent was obtained. Samples were enriched for blasts, and cultured in the presence of SCF, TPO, FLT3-L, IL-3, and IL-6 (KTF36). A panel of 38 drugs was selected from a larger screen of 1839 compounds done on commercially available hematological malignancy cell lines. Drugs included standard chemotherapy agents used in AML and drugs currently under clinical development. Cells were exposed to drugs for 72hrs. An MTS assay was performed and results reported as % of viable cells remaining, after normalization to vehicle control wells. Targeted DNA NGS sequencing of 406 genes, 31 introns, and RNA sequencing of 265 genes was performed for genetic characterization.In vitro drug screening revealed variations in drug sensitivity between samples and revealed time ex-vivo and cytokine milieu to be important factors affecting response of the same cells to the same drugs. Engraftment of long term cultures into immunodeficient mice produced aggressive disease in all cases, indicating robust support of stem cell function via addition of KTF36 cytokines. When possible, clinical response to therapy was compared with in vitro response to the same drugs. This screening approach highlighted well established agents that showed significant activity in highly refractory disease providing rationale for further clinical trial development. An unsupervised clustering showed drug sensitivity primarily correlated with the presence of MLL-X fusion, NRAS/KRAS and PTPN11 alterations. Linear Regression with interaction effects showed drug sensitivity/resistance to be highly selective for single/signature of specific molecular alterations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Predicting Drug Response and Novel Therapeutic Candidates Using Signatures of Molecular Alterations in Hematologic Malignancies

Background: Hematologic malignancies present a varied genetic landscape that plays an important role in prognosis and therapeutic outcome of patients. However, our understanding of the impact of a patient's molecular alterations on therapy is limited. Cell Line-Based High Throughput Screening (CBHTS) can allow novel drug discovery and systematic evaluation of drug response in cell lines or patient samples to large panels of drugs. Here, we present a comprehensive profiling of molecular alterations in hematologic malignancies and their impact on drug response, which we believe provides novel insight into drug sensitivity and resistance correlated with unique molecular alteration signatures as well as clinical trial development.Methods: Cell lines were acquired from ATCC, suspended in growth media, plated in 384 well plates, and allowed to proliferate for 24 hours before addition of drugs. Compounds were obtained from Selleck Chemicals and were added to cells at concentrations of 100nM and 1nM in quadruplicate, with a 0.1% concentration of DMSO. Cell viability was measured after 72 hours with drugs. DMSO was used as the vehicle control, and all drug effects are normalized to the DMSO control and reported as percent viable. Drug activity was studied across a panel of 1,828 drugs and 30 cell-lines. Drug activity clusters were defined using unsupervised learning (Distance metric: Euclidean and Ward linkage; tree height: 1.2). Cell lines were annotated using Broad CCLE and Gnomad database, and mutations were ranked into four groups based on predicted variant effect - high, medium, low, and modifier. Drugs were annotated using Anatomical Therapeutic Classification (ATC), while pathways and targets were annotated using ToppGene Suite. T-statistic was used to estimate significance of differential drug activity between sensitive (cell-lines with viability <20% considered highly sensitive) versus resistant cell-line groups (cell-lines with viability >85% considered highly resistant).Results: We identified 25 clusters with significant differential drug activity across the 30 cell lines. Corticosteroids (n=19), for example, clustered together by activity profile, and showed most differential response across five cell lines: NB4 (Acute Myeloid Leukemia, AML), Kasumi-1 (AML), HL-60 (Acute Lymphoblastic Leukemia, ALL), RS4-11 (ALL) and MV4-11 (Biphenotypic Acute Leukemia, BAL), p = 6.10-27. Steroid activity was minimal in NB4, MV4-11 and HL-60; however, highest in RS4-11 (<11% cell viability) and Kasumi-1. Interestingly, loxapine, an antipsychotic, which acts as a dopamine and serotonin 5-HT2 antagonist, clustered with the main corticosteroid group, and shared similar activity profile (p = 5.5 10-3). A unique gene signature common to RS4-11 and Kasumi-1 included mutations in LOXHD1, FBN3, TRIB3, TDRD6, ALX4, ALDH3B2, NT5DC3, TTC3, ZFAT and GLI2 with moderate variant effects impacting key hematologic processes.Conclusions: This high-dimensional screening against a backdrop of molecular alterations highlighted a potential for correlating differential drug activity with a patient's genetic landscape, with potential for experimental validation and clinical trial development. This pilot study further demonstrates that drug response in hematologic malignancy cell lines may be uniquely driven by a signature of molecular alterations sensitive to single or multiple therapeutic classes and provides rationale for novel drug discovery and drug repositioning in precision medicine. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Probing Distinct Oncogene Addiction By Novel BCL-2 Inhibitors

IntroductionEvading apoptosis is one of the key mechanisms that cells become cancerous. Oncogenic proteins in the family, including BCL-2, BCL-xL, and MCL-1, which counteract the pro-death function of tumor suppressor like proteins, have been shown in many models of tumorigenesis. Thus how to reprogram the apoptosis regulatory machinery to tilt towards pro-death activity has been the focus of modern cancer therapy, regardless of conventional or targeted therapy. Inhibiting BCL-2, BCL-xL and/or MCL-1 anti-death proteins has also become the hot pursuit for drug development. To effectively kill cancers with heterogeneity, it is necessary to understand which oncogene is the underlying mechanism. Applying novel BCL-2 inhibitors, a selective or dual inhibitor for BCL-2/BCL-xL, which have been developed by Ascentage Pharma Group and currently under phase I/II clinical trials, we probed the oncogene addiction patterns in a panel of hematologic malignancy cell lines, including ALL, AML and MM.MethodsCell sensitivity to BCL-2 inhibitors was assessed by Cell-titer-glow assays. 2. Protein expression levels by Western blotting, or protein complexing detected by MSD method were used to dissect the mechanism of sensitivity or resistance. 3. Mice xenograft models were carried out to confirm the tumor growth inhibition (efficacy model) and regression (survival model) through combination treatments. 4. RNAseq was performed to reveal the transcriptome alteration by APG-115, a novel MDM2 inhibitor also developed by Ascentage, to re-sensitize cells to BCL-2 inhibitors.ResultsA panel of hematologic malignancy cell lines were first assessed cells' sensitivity to novel BCL-2/ BCL-xL inhibitors, and revealed a distinct pattern of dependence on BCL-2, BCL-xL or MCL-1, from the most sensitive ALL line RS4;11 to BCL-2, to the most resistant one like MM line H929. In between there is a mix pattern of dependence on these three anti-death proteins.To dissect the mechanism of action, we established and validated BCL-2:BIM complex using MSD method. In BCL-2 dependent cell lines, BCL-2:BIM complex correlates with cells sensitivity to BCL-2 selective inhibitor. While in the mix pattern, high BCL-2:BIM complex alone does not predict drug sensitivity. Pretreating cells with BCL-2 inhibitors, either selective or dual inhibitors, can disrupt the BCL-2:BIM complex and reduce signals detected by MSD methods (Figure 1). Such reduction is consistent with co-immunoprecipitation results using Ascentage selective BCL-2 inhibitor or reference compound ABT-199, suggesting BCL-2 protecting cells through sequestering pro-death protein BIM.To overcome resistance to BCL-2 inhibitor, we combined it with our novel MDM2 inhibitor APG-115. Both in vitro cell viability assay and mice xenograft models demonstrated that MDM2 and BCL-2 inhibitor combination can re-sensitize cells and effectively inhibit tumor growth or lead to tumor regression.To obtain a full transcriptome profile changed by APG-115, we further performed RNAseq on OCI-AML3, which is intrinsically resistant to BCL-2 inhibitor. RNAseq results revealed that APG-115 reprogramed cell cycle and apoptosis pathway, increasing its known targets for cell cycle regulation (p21, p15 protein) and pro-death proteins (BAX, PUMA and NOXA). Furthermore, APG-115 also increased significantly some target genes involved in immune or inflammatory response pathway, including MIC-1 (macrophage inhibitory cytokine-1, GDF15), IL-24 (also known as mda-7, melanoma differentiation-associated protein 7, tumor suppressing protein), and PADI4 (peptidyl arginine deiminase, type IV, regulating granulocyte and macrophage via histone remodeling), which may alter tumor microenvironment and manifest cell death signals (Figure 2).ConclusionsUsing novel selective or dual BCL-2/BCL-xL inhibitor, we probed the oncogene addiction patterns in a panel of hematologic malignancy cell lines, and revealed a mix pattern of dependence on BCL-2, BCL-xL or MCL-1. The BCL-2 addiction relies on the sequestration of pro-death proteins like BIM. Disrupting BCL-2:BIM complex by BCL-2 inhibitors can trigger cell death. MDM2 inhibitor APG-115 reprograms cell cycle, apoptosis and immune/inflammatory pathways to re-sensitize cells to BCL-2 inhibitor. [Display omitted] DisclosuresDeng:Ascentage Pharma Group: Employment. Yin:Ascentage Pharma Group: Employment. Mao:Ascentage Pharma Group: Employment. Zhai:Ascentage Pharma Group: Employment. Yang:Ascentage Pharma Group: Employment. Fang:Ascentage Pharma Group: Employment. Wang:Ascentage Pharma Group: Employment. Zhai:Ascentage Pharma Group Inc.: Employment. Yang:Ascentage Pharma Group Inc.: Employment.

PIM Inhibitor SMI-4a Induces Cell Apoptosis and Cell Cycle Arrest in B-Cell Acute Lymphocytic Leukemia Cells Via the JAK2/STAT3 Pathway

The serine/threonine PIM protein kinases are critical regulators of turmorigenesis in mutiple hematologic malignancies and solid cancers. We used real-time PCR to detect the expression of PIM in B-cell acute lymphocytic leukemia (B-ALL) patients, and found the expression of PIM in B-ALL patients was significantly higher than that in normal controls. SMI-4a is a pan-PIM small molecule inhibitor, and this agent exhibits demonstrable preclinical antitumour activity in a wide range of hematologic malignant cell lines. To further explore the effect of SMI-4a on B-ALL cells, B-ALL cell lines CCRF-SB and Sup-B15 were treated with this small molecule inhibitor, and the results showed that SMI-4a inhibited B-ALL cell proliferation in a dose- and time-dependent manner. Moreover, SMI-4a significantly promoted B-ALL cell apoptosis and caused cell cycle arrest in the G0/G1 phase. The results of Western blot showed that SMI-4a increased the expression of Caspase-3, Caspase-9, Bax and P21, and decreased the expression of Bcl-2 and CDK4. Furthermore, we found that SMI-4a significantly inhibits the activation of the JAK2/STAT3 pathway and HO-1 interferes with the JAK2/STAT3 pathway to inhibit SMI-4a-induced ALL cell apoptosis. Finally, xenograft experiments in NOD/SCID mice were operated to investigate the potential role of SMI-4a in B-ALL tumorigenesis in vivo. To observe the effect of SMI-4a on tumor growth in vivo, NOD/SCID mice were transplanted with B-ALL devied cells, and the tumor-bearing mice were intraperitoneally injected with saline and SMI-4a, respectively. As a result, tretment with SMI-4a resulted in a significant inhibition on tumor growth. In addition, PIM inhibtor obviously reduced the volume and weight of B-ALL cell-derived tumors. TUNEL assay revealed the proportion of apoptotic cells was higher in the SMI-4a-treated group than in the control group. Taken together, our data showed PIM inhibitor (SMI-4a) significantly inhibits the growth of B-ALL cells in vitro and in vivo and promotes apoptosis and cell cycle arrest. This suppressive effect is mediated partly through inhibiting the JAK2/STAT3 pathway activation. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1963: ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma

Abstract Background: B-cell receptor (BCR)-mediated signaling plays an important role in the pathogenesis of a subset of diffuse large B-cell lymphoma (DLBCL). Despite major advances in the treatment, ~40% of the relapsed/refractory DLBCL patients still experience early treatment failure after initial response to chemotherapy. ARQ 531, a reversible inhibitor of BTK and BTK-C481S mutant, also potently suppresses BCR signaling. Here we demonstrate that ARQ 531 targets additional kinases and suppresses multiple oncogenic pathways, this inhibitory potency is coupled to broad anti-tumor activity in DLBCL subtypes including tumors resistant to BCR targeted therapy. Methods: Biochemical inhibition and broad kinase profiling were assessed using recombinant proteins. Binding kinetics and the residence time with BTK and BTK-C481S were measured by Surface Plasmon Resonance (SPR) assay. Binding mode of ARQ 531 with BTK was determined by protein crystallography. Pathway inhibition assessments, in vivo efficacy and in vivo target inhibition were performed in DLBCL tumor models derived from TMD8, SUDHL-4 and DOHH-2 cell lines. Results: ARQ 531 potently inhibited BTK (IC50 = 0.85 nM), the binding potency was accompanied by long residence time (51 min). Crystal structure of BTK/ARQ 531 complex showed that ARQ 531 occupies the ATP-binding pocket. Kinase selectivity profile suggested that ARQ 531 inhibits sub-families of Tec, Src, Trk kinases. Significant anti-proliferative activity (GI50 = &amp;lt; 1µM) was observed in hematological malignant cell lines characterized by addiction to BTK signaling and primarily resistant to ibrutinib. Pathway inhibition analysis suggested that ARQ 531 targets multiple oncogenic signaling pathways in both ABC- and GCB-DLBCL cell lines. Unlike ibrutinib, ARQ 531 suppressed both the upstream activating signals (via inhibition of a select member of Src kinase family) and the downstream signaling pathways (via pAKT and pERK kinases). In GCB-DLBCL cell lines (SUDHL-4 and DOHH-2), ARQ 531 potently suppressed expression of anti-apoptotic c-Myc and BCL6 oncoproteins in a dose dependent fashion, and concomitantly induced apoptotic cleavage of PARP protein. In the ibrutinib-resistant SUDHL-4 mouse xenograft model, ARQ 531 potently suppressed tumor growth (&amp;gt;80% inhibition) compared to the control group when dosed orally at 75 mg/kg. Additionally, preliminary data suggest that ARQ 531 crosses the blood, brain-barrier. Conclusion: ARQ 531 is a potent reversible inhibitor of BTK, its distinct kinase selectivity profile offers significant advantage for simultaneous inhibition of multiple therapeutically relevant targets. ARQ 531 attenuated the expression of key oncogenic drivers via inhibition of downstream BCR activating kinases. These results highlight the therapeutic potential of inhibition of BCR signaling inhibition by ARQ 531 in the treatment of DLBCL. Citation Format: Sudharshan Eathiraj, Yi Yu, Ron Savage, Jennifer A. Woyach, Sean D. Reiff, Amy J. Johnson, Brian Schwartz. ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1963.

Abstract 1877: Selective inhibitor of nuclear export (SINE) compound, eltanexor (KPT-8602), synergizes with venetoclax (ABT-199) to eliminate leukemia cells and extend survival in an in vivo model of acute myeloid leukemia

Abstract SINE compounds (KPT-330/selinexor and KPT-8602/eltanexor) are a novel class of small molecules with therapeutic potential across a broad range of cancers. SINE compounds target exportin 1 (XPO1), which is an essential nucleo-cytoplasmic transport protein, responsible for the nuclear export of &amp;gt;200 cargo proteins including many tumor-suppressor proteins (TSPs) and oncogenes, such as p53, IκB, BCL-2, eIF4E and MYC. XPO1 is overexpressed in numerous solid and hematologic malignancies, often correlating with poor prognosis. An important family of oncoproteins regulated by SINE compounds are the pro-survival BCL-2 family proteins, BCL-2 and MCL-1, as both are transported from the nucleus by forming a complex with the XPO1 cargo protein, elF4E. BCL-2 and MCL-1 are dysregulated and linked to maintenance and survival in numerous cancers. Early studies with venetoclax (VEN/ABT-199), a potent, selective inhibitor of BCL-2, have revealed response rates from 50-80% across a variety of hematologic malignancies. However, resistance often arises via upregulation of other BCL-2 family antiapoptotic proteins, most commonly MCL-1. Thus, we hypothesized that co-treatment of VEN and SINE compounds would show enhanced cell killing in AML due to the ability of SINE to further block the prosurvival signals of both BCL-2 and MCL-1. Co-treatment with eltanexor and VEN demonstrated synergistic loss of viability in multiple hematologic malignancy cell lines, particularly MV-4-11 cells. Further in vitro analysis showed this enhanced cell death was due to an increase in apoptosis that led to a loss of clonogenicity in methylcellulose assays. We used in vivo studies to test the effect of this combination in a systemic AML xenograft model. For this, MV-4-11 cells were transplanted into NSGS mice and allowed to engraft before treating the mice with vehicle, eltanexor, VEN, or a combination of the two compounds. When the vehicle mice became moribund at 4 weeks after transplant, all treatment groups were sacrificed to compare leukemic burden in the hematopoietic tissues. Flow cytometric analysis revealed a drastic elimination of leukemic cells in the bone marrow, spleen, and peripheral blood of the combination-treated mice compared to vehicle and single-treatment groups. Immunohistochemical analysis of the spleen and bone marrow confirmed this finding, and also revealed the reduction in leukemic cells was due, in part, to an induction of apoptosis (an increase in cleaved caspase 3 and decrease in survivin) in splenic tissue sections, in agreement with the in vitro results. Finally, the dramatic clearance of leukemia cells in the mouse caused by eltanexor and VEN treatment led to prolonged survival. Our results demonstrate the potential of this combination regimen in the treatment of AML. Citation Format: Melissa A. Fischer, Pia M. Arrate, Hua Chang, Agnieszka E. Gorska, Londa S. Fuller, Haley E. Ramsey, Trinayan Kashyap, Christian Argueta, William Senapedis, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Michael R. Savona. Selective inhibitor of nuclear export (SINE) compound, eltanexor (KPT-8602), synergizes with venetoclax (ABT-199) to eliminate leukemia cells and extend survival in an in vivo model of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1877.

Identification of Selective, Cell Active Inhibitors of Protein Arginine Methyltransferase 5 through Structure-Based Virtual Screening and Biological Assays.

Protein arginine methyltransferase 5 (PRMT5), a type II PRMT enzyme, is reported as an important therapeutic target in leukemia and lymphoma. In the present study, based on the combination of virtual screening and biochemical validations, we discovered a series of small-molecule inhibitors targeting PRMT5. Among those, DC_Y134 exhibited the most potent activity with IC50 value of 1.7 μM and displayed good selectivity against other methyltransferases. Further treatment with DC_Y134 inhibited the proliferation of several hematological malignancy cell lines by causing cell cycle arrest and apoptosis. Western blot assays indicated that DC_Y134 reduced the cellular symmetrically dimethylated levels. In addition, we analyzed the binding mode of DC_Y134 through molecular docking, which revealed that DC_Y134 occupies the binding site of substrate arginine and explained the selectivity of this inhibitor. Taken together, compound DC_Y134 could be used to elucidate the biological roles of PRMT5 and serve as a lead compound for treatment of hematologic malignancies.

Effects of human umbilical cord-derived mesenchymal stem cells on hematologic malignancies.

Mesenchymal stem cells (MSCs) have been used in hematopoietic stem cell transplantation for years. However, the safety of MSCs applied in various types of hematologic malignancy has not been comprehensively explored. In the present study, the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on six representative hematologic malignancy cell lines were explored, including leukemia, multiple myeloma and lymphoma cells. Direct and indirect co-culture models were established, and cell proliferation was assessed by carboxyfluorescein diacetate succinimidyl ester staining. A cytometric bead array cytokine kit was used to quantify cytokines. The expression of interleukin (IL)-6 receptor elements on tumor cells was detected by reverse transcription-polymerase chain reaction and flow cytometry, and the effects of exogenous IL-6 on cell proliferation were determined using a Cell Counting kit-8 assay. The results demonstrated that hUC-MSCs inhibited the proliferation of most of the cell lines examined (THP-1, HL-60, K562 and RPMI-8226), but promoted the proliferation of Raji cells. In addition, hUC-MSCs secreted abundant IL-6, promoted the secretion of IL-10 by RPMI-8226 and Raji cells, and inhibited the secretion of tumor necrosis factor-α by THP-1 cells. These data indicate a varied effect of hUC-MSCs on various types of hematologic malignancy, including distinct mechanisms of cell-to-cell contact and cytokines. Researchers applying hUC-MSCs in lymphoma should be aware of a potential tumor growth-promoting effect.

Abstract 44: CG′806, a first-in-class FLT3/BTK inhibitor, exhibits potent activity against AML patient samples with mutant or wild type FLT3, as well as other hematologic malignancy subtypes

Abstract The Fms-like receptor tyrosine kinase 3 (FLT3) is expressed in approximately 90% of acute myeloid leukemia (AML), almost all B-lineage acute lymphoblastic leukemia (B-ALL), and some other hematologic malignancy subtype patients. Approximately one third of AML patients harbor constitutive activating internal tandem duplication in FLT3 (FLT3-ITD), which is associated with very poor prognosis. These observations have made FLT3 an attractive drug target. As more potent FLT3 inhibitors are developed, additional acquired point mutations have been identified, commonly at activation loop residue D835 and “gatekeeper” residue F691, which are related to drug resistance and relapse. CG′806, a first-in-class small molecule multikinase inhibitor against FLT3 and Bruton's tyrosine kinase (BTK), is under development as a next-generation agent for the treatment of FLT3-driven AML. Work from our collaborative laboratories has demonstrated that CG′806 retains full activity against Ba/F3 cells housing FLT3-ITD and/or point mutations, including D835G, D835Y, D835H, and F691L (abstract submitted to 2017 AACR Hematologic Malignancy Meeting). Herein we evaluated the potency of CG′806 on various hematologic malignancy cell lines and patient primary bone marrow specimens. Primary patient mononuclear cells were derived from 172 patients diagnosed with AML (n=82), myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPN, n=15), ALL (n=17), and chronic lymphocytic leukemia (CLL, n=58). Sensitivity to CG′806 was evaluated across a range of concentrations after 72-hour treatment. IC50s were calculated as a measure of drug sensitivity and compared to nonproprietary competitive agents in AML and CLL, including the bromodomain inhibitors OTX-015 and JQ-1 and the FLT3 inhibitor quizartinib. The inhibitory effect of CG′806 on FLT3 signaling was evaluated in MV4-11 cells by Western blotting. Across the four general subtypes of hematologic malignancies in the dataset, there is broad sensitivity to CG′806 with 59% (48/82) AML, 29% (5/17) ALL, 53% (8/15) MDS/MPN, and 40% (23/58) CLL cases exhibiting an IC50 of less than 0.1 μM. CG′806 demonstrated median IC50s of 0.07 μM and 0.22 μM against primary AML and CLL cells, respectively. FLT3 mutational status is known for 38 of the tested AML patient samples. Among those, the FLT3-ITD+ AML samples tended to have enhanced sensitivity to CG′806 (median IC50 = 0.02 μM, n=8) as compared to the FLT3-WT samples (median IC50 = 0.12 μM, n=30). CG′806 also exerted potent picomolar to low nanomolar IC50 antiproliferative activity against human AML, B-ALL, mantle cell lymphoma, Burkitt's lymphoma, and diffuse large B-cell lymphoma cell lines. As compared to the FLT3 inhibitor quizartinib, CG′806 at a concentration of 500 pM completely inhibited phosphorylation of FLT3 and STAT5 in MV4-11 cells upon 1 hour treatment, whereas quizartinib only partially inhibited their phosphorylation. CG′806 exhibits broad and potent activity against AML patient samples, as well as other hematologic malignancy subtypes. The median IC50 data for CG′806 indicates greater potency relative to the tested nonproprietary agents across AML and CLL. Preliminary analyses reveal a trend of greater sensitivity to CG′806 in FLT3 mutant AML cases compared with FLT3 wild type; however, ongoing accrual of additional patient samples will be required to sufficiently power a statistical association of CG'806 sensitivity with FLT3 mutational status. In summary, preclinical analyses of CG′806 against primary hematologic malignancy patient samples and cultured cell lines show evidence of broad drug activity in AML and other disease subtypes and support further development of this agent for hematologic malignancies. Citation Format: Stephen E. Kurtz, Beth Wilmot, Shannon McWeeney, Avanish Vellanki, Andrea Local, Khalid Benbatoul, Peter Folger, Susan Sheng, Hongying Zhang, Stephen B. Howell, William G. Rice, Brian J. Druker, Jeffrey W. Tyner. CG′806, a first-in-class FLT3/BTK inhibitor, exhibits potent activity against AML patient samples with mutant or wild type FLT3, as well as other hematologic malignancy subtypes [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 44.

Novel pan PI3K inhibitor-induced apoptosis in APL cells correlates with suppression of telomerase: An emerging mechanism of action of BKM120

The intertwining between cancer pathogenesis and perturbation of multitude signaling pathways ushered the cancer therapeutic approaches into an unbounded route of targeted therapies. For the nonce and among the plethora of promising inhibitors, intense interest has focused on small molecules targeting different component of PI3K axis. Intrigued by the constant activation of PI3K in leukemia, this study aimed to investigate the effects of BKM120, as the excelled member of pan PI3K inhibitors, in a panel of hematologic malignant cell lines. The resulting data showed that BKM120 exerted a concentration-dependent growth suppressive effect; however, IC50 values varied among the tested cells. Our results outlined that the blockage of PI3K in NB4, as the most sensitive cell line, resulted in a caspase-3-dependent apoptosis probably through NFκB-mediated suppression of c-Myc and hTERT. As far we are aware, to date, there have been no reports of BKM120 effect on enzymatic repression of telomerase, and this study represents for the first time that the anti-proliferative effect of the inhibitor on NB4 is mediated by down-regulation of telomerase; shedding new light on the novel mechanism of action of BKM120.

Abnormal Expression of the Chemokine (C-C Motif) Ligand 17 Gene in Adult B-Cell Acute Lymphoblastic Leukemia

AbstractBackground: Chemokine (C-C Motif) Ligand 17 is a protein coding gene. This chemokine plays important roles in T cell development in thymus as well as in trafficking and activation of mature T cells. Diseases associated with CCL17 include mycosis fungoides, paragonimiasis and some hematologic malignancies such as Hodgkin¡¯s lymphoma, B cell lymphoma, and Nasal natural killer/T cell lymphoma (NNKTL). However, our knowledge of the expression levels of CCL17 in B-cell acute lymphoblastic leukemia (ALL) remains limited.Aims: The purpose of this study was to investigate the expression levels of human CCL17 messenger RNA in adult B-cell ALL.Methods: A real-time quantitative reverse transcription-polymerase chain reaction assay based on TaqMan fluorescence methodology was used to quantify the CCL17 mRNA copy number in the bone marrow cells from patients with adult leukemia and in 16 human hematologic malignant cell lines. Normal marrow samples from the allogeneic stem cell transplantation donors were served as control. Informed consent was obtained for every marrow sample.Results:Expression levels of the CCL17 gene in leukemic cell lines, leukemia patients and normal donor marrow are shown in Figure 1. These results showed that the relative levels of CCL17 gene expression in marrow from 189 newly diagnosed B-cell ALL(median 0.23%; range 0%¡«69000%) was significantly higher than those of bone marrow from the 43 healthy donors (median 0.05%; range 0%¡«6.87%;P =0.0007). Significant CCL17 mRNA overexpression was found in the de novo B-cell ALL patients compared with 102 treated B-cell ALL patients(median 0.02%; range 0%¡«3339%) who achieved complete remission or 70 de novo AML (median 0.02%; range 0%¡«9.132%;P¡¯s <0.0001). The expression levels of CCL17 was higher in 21 refractory/relapsed B-cell ALL patients (median 0.68%; range 0.003%¡«13490%) than that newly diagnosed B-cell ALL, but no statistical significant difference was observed (P=0.34). Besides, no statistical significant difference was observed in 16 newly diagnosed T-cell ALL(median 0.06%, range 0%¡«9.947%), 70 AML and 43 healthy donors (P¡¯s > 0.05), but it was higher in SupB15 and Nalm-6 cells from B-cell ALL cell lines than in other cells from AML or T-cell ALL cell lines.Conclusion: These results suggest that abnormal expression of CCL17 in leukemia may be involved in the pathomechanism of B-cell ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gene expression profiling of hematologic malignant cell lines resistant to oncolytic virus treatment.

Pexa-Vec (pexastimogene devacirpvec; JX-594) has emerged as an attractive tool in oncolytic virotherapy. Pexa-Vec demonstrates oncolytic and immunotherapeutic mechanisms of action. But the determinants of resistance to Pexa-Vec are mostly unknown. We treated hemoatologic malignant cells with Pexa-Vec and examined the gene-expression pattern of sensitive and resistant cells. Human myeloid malignant cell lines (RPMI-8226, IM-9, K562, THP-1) and lymphoid cancer cell lines (MOLT4, CCRF-CEM, Ramos, U937) were treated with Pexa-Vec. Pexa-Vec was cytotoxic on myeloid cell lines in a dose-dependent manner, and fluorescent imaging and qPCR revealed that Pexa-Vec expression was low in RAMOS than IM-9 after 24 hrs and 48 hrs of infection. Gene expression profiles between two groups were analyzed by microarray. Genes with at least 2-fold increase or decrease in their expression were identified. A total of 660 genes were up-regulated and 776 genes were down-regulated in lymphoid cancer cell lines. The up- and down-regulated genes were categorized into 319 functional gene clusters. We identified the top 10 up-regulated genes in lymphoid cells. Among them three human genes (LEF1, STAMBPL1, and SLFN11) strongly correlated with viral replication. Up-regulation of PVRIG, LPP, CECR1, Arhgef6, IRX3, IGFBP2, CD1d were related to resistant to Pexa-Vec. In conclusion, lymphoid malignant cells are resistant to Pexa-Vec and displayed up-regulated genes associated with resistance to oncolytic viral therapy. These data provide potential targets to overcome resistance, and suggest that molecular assays may be useful in selecting patients for further clinical trials with Pexa-Vec.

Expression and Subcellular Distribution of Costimulatory Molecules B7-H1,B7-H3 and B7-H4 in Human Hematologic Malignancy Cell Lines

To investigate the expression and subcellular distribution of costimulatory molecules B7-H1, B7-H3 and B7-H4 in human hematologic malignancy cell lines. The expression and subcellular distribution of B7-H1, B7-H3 and B7-H4 in 13 human hematologic malignancy cell lines were determined by RT-PCR, qPCR, Western blot and flow cytometry, the peripheral blood mononuclear cells (PB MNC) of 12 volunteers were used as control. The mRNA of B7-H1, B7-H3 and B7-H4 was widely expressed in PB MNC and hematologic malignancy cell lines, with a lower level of B7-H4. The mRNA expression of 3 molecules was highest in Maver, Z138, and HL-60, respectively, while among them the B7-H3 and B7-H4 had no expression in CZ1. The nuclear and cytoplasmic protein of 3 costimulatory molecules abnormally overexpressed only in hematologic malignancy cell lines, with the highest level in U937, Z138, and Raji, respectively, while the B7-H3 and B7-H4 had no expression in CZ1. There were differences among mRNA expression, nuclear and cytoplasmic protein expression of 3 molecules in cell lines derived from the same type of tumor, but the differences of expression in mRNA and protein levels were not exactly the same. The B7-H3 expression abundance in membrane localization was higher in U937, Maver and Z138, while the membrane protein of B7-H1 and B7-H4 had no or low expression in 13 cell lines. The mRNA expression of costimulatory molecules B7-H1, B7-H3 and B7-H4 can be widely detected. The protein level of 3 costimulatory molecules abnormally overexpressed only in hematologic malignancy cell lines, moreover the subcellular localizations mostly was found in nucleus and cytoplasm, while the membrane protein expresses in low level or had no expression. There are differences among the expression of 3 molecules in cell lines derived from the same type of tumor.

B7-H3 Silencing By RNAi Inhibits Tumor Progression and Enhances Chemosensitivity in U937 Cells

Background: Aberrant expression of the immunoregulatory protein B7-H3 in B7 family has been associated with more advanced disease and poor prognosis in a wide range of cancer. However, the role of B7-H3 in acute monocytic leukemia U937 cells has not been thoroughly investigated. In this study, we examined the gene expression and subcellular localization of B7-H3 in human peripheral blood cells and hematologic malignancy cell lines. Then, we determined the effects of down-regulating B7-H3 expression on U937 cells, which has the highest B7-H3 protein expression in the detected cell lines.Methods: B7-H3 expression in 12 healthy volunteer peripheral blood cell samples and 13 human hematologic malignancy cell lines was determined by RT-PCR, western blot and flow cytometry. B7-H3 knockdown in the U937 cell line was performed using shRNA lentivirus transduction. The effects on cell proliferation, cycle, migration and invasion were investigated by CCK-8 assay, methyl cellulose colony forming assay, PI staining, and transwell assays in vitro. U937 xenograft models were used to assess the effects of B7-H3 on tumorigenicity and Ki-67 and PCNA was detected through immunohistochemical. Changes in cell growth inhibition and apoptosis, when combined with chemotherapy drugs, were determined using CCK-8, Annexin V-FITC/PI and Hoechst 33342 staining assays in vitro. The therapeutic effect of B7-H3 knockdown in combination with chemotherapy drugs were also studied by U937 xenograft models in vivo.Results: B7-H3 mRNA was widely expressed in the 12 hematologic malignancy cell lines except for CZ1 and PB MNCs of volunteers. But the protein level of B7-H3 was only abnormally overexpressed in 12 hematologic malignancy cell lines except for CZ1, with subcellular localizations in nucleus and cytoplasm mostly determined. The down-regulation of B7-H3 in U937 cells significantly decreased cell growth and the rate of colony formation by 32.8% in 72 h and 70.3% in 14 d. Mean inhibition rate of tumor growth with B7-H3 knockdown was 59.4%, and expression of both Ki-67 and PCNA in xenografts was significantly reduced. After B7-H3 silencing, U937 cell cycle was arrested at G0/G1 phase, and the cell cycle-related proteins Cyclin D1 and CDK4 were lower. Cell migration rate of B7-H3 knockdown cells was reduced more than five-fold, and invasion capacity was decreased by 86.7%. The rates of distant metastasis in B7-H3 knockdown xenografts were significantly decreased. The invasion-related proteins MMP-2 and MMP-9 were lower in both B7-H3 knockdown cells and xenografts. B7-H3 RNAi profoundly increased the anti-tumor effect of chemotherapy and enhanced the activity of caspase-3 in vitro and in vivo. At the end of observation (on day 19 after inoculation), inhibition rates of tumor growth in B7-H3 shRNA combined with idarubicin, cytarabine, and idarubicin plus cytarabine groups were 70.5%, 80.0%, and 90.0%, respectively (P =0.006, 0.004 and 0.016). The TUNEL positive cells were significantly increased in the B7-H3 shRNA combined with chemotherapy drugs groups.Conclusions: B7-H3 protein was abnormally overexpressed in 12 hematologic malignancy cell lines except for CZ1, with subcellular localizations in nucleus and cytoplasm mostly determined. B7-H3 may promote U937 cell progression, and shRNA targeting B7-H3 significantly enhances sensitivity to chemotherapeutic drugs. These results may provide new insight into the function of B7-H3 and a promising therapeutic approach targeting B7-H3 in acute monocytic leukemia. DisclosuresNo relevant conflicts of interest to declare.

Effects of Garcinia Acid Combined with Daunorubicin on Expression of Pregnane X Receptor in Leukemia Cell Line K562/A02

To explore the expression of PXR (Pregnane X receptor) in several malignant hematological cell lines, and to investigate the reversal effect of Gambogic acid (GA) on multi-drug resistance (MDR) of K562/A02 cell line and its reversal mechanism. Transcription of PXR was detected by real-time PCR in several malignant hematological cell lines. The growth inhibition rate of K562/A02 in different experimental groups was assayed by MTT method, and the expression of PXR protein was measured by Western blot. PXR gene transcription could be detected in several hematological malignancy cell lines, and it was significantly higher in K562/A02 cell line, compared with the other cell lines used in this experiment. Low-dose GA could enhance cell growth inhibition rate, increasing the effect of chemotherapy, which may be associated with down-regulation of PXR expression. PXR gene transcription and protein expression in GA and DNR+GA groups decreased as compared with control group and the DNR group, suggesting that low-dose GA can down-regulate PXR gene transcription and protein expression. PXR gene transcription can be detected in several hematological malignancy cell line, which is significantly higher in K562/A02 cell line, as compared with the other cell lines used in this experiment. Low-dose GA can enhance cell growth inhibition rate, increasing the effect of chemotherapy, which may be associated with down-regulation of PXR expression.

Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS).

BackgroundDNA methylation has essential roles in transcriptional regulation, imprinting, X chromosome inactivation and other cellular processes, and aberrant CpG methylation is directly involved in the pathogenesis of human imprinting disorders and many cancers. To address the need for a quantitative and highly multiplexed bisulfite sequencing method with long read lengths for targeted CpG methylation analysis, we developed single-molecule real-time bisulfite sequencing (SMRT-BS).ResultsOptimized bisulfite conversion and PCR conditions enabled the amplification of DNA fragments up to ~1.5 kb, and subjecting overlapping 625–1491 bp amplicons to SMRT-BS indicated high reproducibility across all amplicon lengths (r = 0.972) and low standard deviations (≤0.10) between individual CpG sites sequenced in triplicate. Higher variability in CpG methylation quantitation was correlated with reduced sequencing depth, particularly for intermediately methylated regions. SMRT-BS was validated by orthogonal bisulfite-based microarray (r = 0.906; 42 CpG sites) and second generation sequencing (r = 0.933; 174 CpG sites); however, longer SMRT-BS amplicons (>1.0 kb) had reduced, but very acceptable, correlation with both orthogonal methods (r = 0.836-0.897 and r = 0.892-0.927, respectively) compared to amplicons less than ~1.0 kb (r = 0.940-0.951 and r = 0.948-0.963, respectively). Multiplexing utility was assessed by simultaneously subjecting four distinct CpG island amplicons (702–866 bp; 325 CpGs) and 30 hematological malignancy cell lines to SMRT-BS (average depth of 110X), which identified a spectrum of highly quantitative methylation levels across all interrogated CpG sites and cell lines.ConclusionsSMRT-BS is a novel, accurate and cost-effective targeted CpG methylation method that is amenable to a high degree of multiplexing with minimal clonal PCR artifacts. Increased sequencing depth is necessary when interrogating longer amplicons (>1.0 kb) and the previously reported bisulfite sequencing PCR bias towards unmethylated DNA should be considered when measuring intermediately methylated regions. Coupled with an optimized bisulfite PCR protocol, SMRT-BS is capable of interrogating ~1.5 kb amplicons, which theoretically can cover ~91% of CpG islands in the human genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1572-7) contains supplementary material, which is available to authorized users.

Effects of Resveratrol and Vitamin C on the Self-Renewal Capacity of Blast Progenitors in Hematologic Malignancies

Although a large number of phytochemicals has been shown to inhibit the growth of leukemic and cancer cells, their effects on the self-renewal (SR) capacity of the malignant stem/progenitor cells were not examined. In seven hematologic malignant cell lines including U-937, Mo7e, K-562, HL-60, U-266, Raji and Daudi, the SR capacity of blast progenitors was assessed by measuring cumulative clonogenic cells in suspension, whereas the terminal division of blast progenitors was assessed by blast colony formation (BCF) in methylcellulose. Resveratrol and vitamin C (Vit-C) with mean IC50 values of 41 and 514 μM for inhibiting BCF were added twice per week to cells in long-term suspension at 10 and 300 μM, respectively. Vit-C induced slight increase in senescence or necrosis rates, marked G2/M arrest and significant inhibition of telomerase activity in two, one and five cell lines, respectively. Resveratrol, but not Vit-C, induced cell cycle arrest and/or cellular stress responses such as senescence, apoptosis, and necrosis and inhibited SR in all seven cell lines. Resveratrol coordinately induced S-phase arrest and cellular stress responses, inhibited telomerase activity and abolished SR in U-937, Mo7e and K-562 cells. Resveratrol induced marked increase in p21 and/or p27 transcript levels and senescence rates in U-937, K-562 and U-266 cells. Taken together, resveratrol has been shown to exert multifactorial actions and inhibit SR, whereas Vit-C by inhibition of telomerase activity alone did not affect SR.

Abstract 1805: Characterization of pyoluteorin derivatives as Mcl-1 antagonists

Abstract The evasion of apoptosis is considered to be a hallmark of cancers and a cause of resistance to anti-cancer therapies. Consistently, aberrant overexpression of anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-xL, Mcl-1) is associated with tumorigenesis and increased resistance to chemotherapy in multiple malignancies. To date, several small-molecule Bcl-2 inhibitors that bind the BH3-binding pocket of the protein have entered clinical trials. These BH3 mimetics are known to induce apoptosis by disrupting the interactions between pro- and anti-apoptotic Bcl-2 family proteins. The most well characterized selective agents are the Bcl-2 inhibitor ABT-737 and its orally active analog ABT-263 which inhibit Bcl-2 and Bcl-XL but not Mcl-1. Consequently, cancers with elevated Mcl-1 do not respond to these drugs. Moreover, Mcl-1 is frequently upregulated in cancer cells and compensates for the inhibition of Bcl-2 function by ABT-737, thereby mediating resistance to the drug. Thus, there is an urgent need to develop Mcl-1-specific inhibitors for the treatment of Mcl-1-dependent human cancers. We have recently identified and characterized the natural product marinopyrrole A (maritoclax) as a novel Mcl-1 inhibitor, which induces apoptosis by targeting Mcl-1 for proteasomal degradation without significant effects on mRNA levels. To determine the pharmacophore of maritoclax, we systematically altered or removed portions of the molecule to identify regions essential for inhibition of Mcl-1 and induction of apoptosis. To date, we synthesized over 40 derivatives of maritoclax and evaluated their inhibitory actions toward Mcl-1 and cytotoxicity in cell lines of human hematological malignancies. As a result, we have identified several pharmacophore sites in maritoclax that are required for biological activity and two pyoluteorin derivatives, KS04 and KS18, which bind to a similar site(s) in Mcl-1 as maritoclax, as determined by NMR titration experiments. Like maritoclax, KS04 or KS18 markedly reduced the half-life of Mcl-1 protein, induced apoptosis selectively in Mcl-1-dependent but not Bcl-2-dependent K562 cells, and acted synergistically with ABT-737 to enhance apoptosis in ABT-737 resistant HL60 cells. Moreover, the combination treatment of KS18 (10 mg/kg/d) and ABT-737 (20 mg/kg/d) significantly suppressed the growth of ABT-737-resistant HL60 xenografts in nude mice. Thus, further studies are warranted to develop pyoluteorin derivatives as a novel class of Mcl-1 inhibitors. Citation Format: Kenichiro Doi, Krishne Gowda, Qiang Liu, Shen-Shu Sung, Jyh Ming Lin, Shantu Amin, Thomas P. Loughran, Hong-Gang Wang. Characterization of pyoluteorin derivatives as Mcl-1 antagonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1805. doi:10.1158/1538-7445.AM2014-1805

Neuroimaging of Movement Disorders

1389 Objectives Cancer cell lines, established from either primary or metastatic sites of cancer patients, have contributed to understanding cancer biology and the selection/validation of imaging radiotracers. Cell lines with a shorter doubling time (DT) are expected to have more aggressive behavior. In the present study, we assessed correlations between the DTs of hematological malignant cell lines and the abundance of messages for Ki67, PCNA, TK1, thymidylate synthase (TYMS), HK1, or transporters (SLC29A1-2) Methods A NCI 60 exon dataset (interrogating over 1 million exon clusters within the known and predicted transcribed regions of the entire genome) was selected from NIH Gene Expression Omnibus repository, which contains 178 chips including 6 hematological malignant cell lines with triplicates for each: HL-60, RPMI-8226, K-562, SR, CCRF-CEM, and MOLT-4. Using Partek, commercial software, we obtained an average abundance of the transcripts of interest above. Correlations between the abundance of genes and NCI documented doubling times were determined by Pearson correlation testing (α=0.05 for a one-tailed test). Results The correlation coefficients r of HK1 message (phosphorylating FDG) vs. DTs was -0.87 (p= 0.01 Conclusions In these cell lines, HK1 message abundance appeared to have a stronger correlation to DTs of hematological malignant cancer cell lines than message levels for TK1, TYMS, and conventional proliferation markers Ki67 and PCNA. The correlation is negative, supporting higher HK1 abundance and shorter DT. Since message levels and protein levels can sometimes be disparate, our findings should be validated using protein quantitation and in vivo. Research Support NIH T32 training grant