Cell Line KU812 Research Articles

Abstract 521: Anticancer mechanisms of epigallocatechin gallate revealed via cellular and molecular profiling

Abstract Introduction: Epigallocatechin gallate (EGCG) is a polyphenol present in green tea that is known for its anti-oxidant, anti-inflammatory, anti-angiogenic, pro-apoptotic, and anti-cancer properties. However, EGCG has not been systematically evaluated on a large panel of cancer cell lines. We hypothesized that EGCG exhibits varying effects on specific cancer cell genetic backgrounds and types. This study aimed to provide a comprehensive analysis of EGCG on both cell viability and gene expression profiles. Methods: 750 cancer cell lines were cultured and seeded in 1536-well plates for cell viability studies (by the CTD2 Center at the Broad Institute). After 24 h, cells were treated with 16 two-fold serial dilutions of EGCG. After 72 h, cell viability was assayed using CellTiter-Glo. For gene expression profiling (with the Broad Institute and NIH), 8 cell lines were treated with 10 µM EGCG for 6 and 24 h, and transcriptomes were assessed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. The CMap large-scale transcriptome dataset was used for comparisons. Results: EGCG had the highest potency in the non-Hodgkins B-cell lymphoma cell line DoHH2 (IC50=0.42 µM), the myeloid leukemia cell line Ku812 (IC50=3.76 µM), and the multiple myeloma cell line KMS-28BM (IC50=4.57 µM). Tissue-of-origin analysis showed that EGCG was most potent against lymphoid cancers, and least potent against prostate cancers. Bioinformatics-based analyses found that EGCG-treated cells resembled cells overexpressing CHEK2, DDP4, CBLC, ZNF350, and TRAF6, and cells underexpressing TRIM16. EGCG-induced gene expression resembled lapatinib and sulfasalazine treatment. Conclusions: This study provides a comprehensive examination of the differential impact of EGCG treatment on cancer cells. Subsequent research on the timing and duration of the changes, along with pathway enrichment analyses, will potentially guide the translational potential of EGCG and its chemical analogs in personalized cancer treatment approaches. Citation Format: Aria Panchal, Jacqueline H. Law, Clement Lo, Kenneth W. Yip. Anticancer mechanisms of epigallocatechin gallate revealed via cellular and molecular profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 521.

CAR virus receptor mediates erythroid differentiation and migration and is downregulated in MDS.

Myelodysplastic syndromes (MDS) are myeloid neoplasms presenting with dysplasia in the bone marrow (BM) and peripheral cytopenia. In most patients anemia develops. We screened for genes that are expressed abnormally in erythroid progenitor cells (EP) and contribute to the pathogenesis of MDS. We found that the Coxsackie-Adenovirus receptor (CAR = CXADR) is markedly downregulated in CD45low/CD105+ EP in MDS patients compared to control EP. Correspondingly, the erythroblast cell lines HEL, K562, and KU812 stained negative for CAR. Lentiviral transduction of the full-length CXADR gene into these cells resulted in an increased expression of early erythroid antigens, including CD36, CD71, and glycophorin A. In addition, CXADR-transduction resulted in an increased migration against a serum protein gradient, whereas truncated CXADR variants did not induce expression of erythroid antigens or migration. Furthermore, conditional knock-out of Cxadr in C57BL/6 mice resulted in anemia and erythroid dysplasia. Finally, decreased CAR expression on EP was found to correlate with high-risk MDS and decreased survival. Together, CAR is a functionally relevant marker that is down-regulated on EP in MDS and is of prognostic significance. Decreased CAR expression may contribute to the maturation defect and altered migration of EP and thus their pathologic accumulation in the BM in MDS.

Discovery of CZS-241: A Potent, Selective, and Orally Available Polo-Like Kinase 4 Inhibitor for the Treatment of Chronic Myeloid Leukemia

Recent studies demonstrate that PLK4 has emerged as a therapeutic target for the treatment of multiple cancers owing to its indispensable role in cell division. Herein, starting from previously identified effective compound CZS-034, based on rational drug design strategies, tyrosine kinase receptor A (TRKA) selectivity- and metabolic stability-guided structure-activity relationship (SAR) exploration were carried out to discover a highly potent (IC50 = 2.6 nM) and selective (SF = 1054.4 over TRKA) PLK4 inhibitor B43 (CZS-241) with acceptable human liver microsome stability (t1/2 = 31.5 min). Moreover, compound B43 effectively inhibited leukemia cells in 29 tested cell lines, especially chronic myeloid leukemia (CML) cell lines K562 and KU-812. Pharmacokinetic characteristics revealed that compound B43 possessed over 4 h of half-life and 70.8% bioavailability in mice. In the K562 cells xenograft mouse model, a 20 mg/kg/day dosage treatment obviously suppressed tumor progression. As a potential and novel PLK4-targeted candidate drug for CML, compound B43 is undergoing extensive preclinical safety evaluation.

AMPK inhibition induces MCL1 mRNA destabilization via the p38 MAPK/miR-22/HuR axis in chronic myeloid leukemia cells

The oncogenic and tumor-suppressive roles of AMPK in chronic myeloid leukemia (CML) are controvertible. This study aimed to investigate the cytotoxic effects of the AMPK inhibitor Compound C in the CML cell lines K562, KU812, and MEG-01. Compared to K562 cells, KU812 and MEG-01 cells were more sensitive to Compound C-mediated cytotoxicity. Moreover, Compound C induced SIRT3 upregulation in K562 cells but not in KU812 or MEG-01 cells. SIRT3 silencing increased the sensitivity of K562 cells to Compound C. Additionally; Compound C-induced autophagy attenuated its induced apoptosis in KU812 and MEG-01 cells. Compound C-induced ROS-mediated AMPKα inactivation resulted in the downregulation of apoptotic regulator MCL1 in KU812 and MEG-01 cells. Mechanistically, AMPK inhibition activated p38 MAPK-mediated miR-22 expression, which in turn inhibited HuR expression, thereby reducing MCL1 mRNA stability. Overexpression of constitutively active AMPKα1 and abolishment of the activation of p38 MAPK inhibited Compound C-induced cell death and MCL1 downregulation. Furthermore, Compound C synergistically enhanced the cytotoxicity of BCR-ABL inhibitors and the BCL2 inhibitor ABT-199. Collectively, this study indicates that Compound C induces MCL1 downregulation through the AMPK/p38 MAPK/miR-22/HuR pathway, thereby inducing apoptosis of KU812 and MEG-01 cells. Furthermore, our findings suggest that AMPK inhibition is a promising strategy for improving CML therapy.

Exposure of the Basophilic Cell Line KU812 to Liposomes Reveals Activation Profiles Associated with Potential Anaphylactic Responses Linked to Physico-Chemical Characteristics.

Lipidic nanoparticles (LNP), particularly liposomes, have been proven to be a successful and versatile platform for intracellular drug delivery for decades. Whilst primarily developed for small molecule delivery, liposomes have recently undergone a renaissance due to their success in vaccination strategies, delivering nucleic acids, in the COVID-19 pandemic. As such, liposomes are increasingly being investigated for the delivery of nucleic acids, beyond mRNA, as non-viral gene delivery vectors. Although not generally considered toxic, liposomes are increasingly shown to not be immunologically inert, which may have advantages in vaccine applications but may limit their use in other conditions where immunological responses may lead to adverse events, particularly those associated with complement activation. We sought to assess a small panel of liposomes varying in a number of physico-chemical characteristics associated with complement activation and inflammatory responses, and examine how basophil-like cells may respond to them. Basophils, as well as other cell types, are involved in the anaphylactic responses to liposomes but are difficult to isolate in sufficient numbers to conduct large scale analysis. Here, we report the use of the human KU812 cell line as a surrogate for primary basophils. Multiple phenotypic markers of activation were assessed, as well as the release of histamine and inflammasome activity within the cells. We found that larger liposomes were more likely to result in KU812 activation, and that non-PEGylated liposomes were potent stimulators of inflammasome activity (four-fold greater IL-1β secretion than untreated controls), and a lower ratio of cholesterol to lipid was also associated with greater IL-1β secretion ([Cholesterol:DSPC ratio] 1:10; 0.35 pg/mL IL-1β vs. 5:10; 0.1 pg/mL). Additionally, PEGylation appeared to be associated with direct KU812 activation. These results suggest possible mechanisms related to the consequences of complement activation that may be underpinned by basophilic cells, in addition to other immune cell types. Investigation of the mechanisms behind these responses, and their impact on use in vivo, are now warranted.

Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals.

CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we discovered a novel rat monoclonal antibody #147D. This humanized IgG4-formatted antibody, h4#147D, showed potent antitumor efficacy in xenograft mouse models harboring the human PDAC cell line MIA PaCa-2, HCC cell line Hep G2, and CML cell line KU812, which featured low sensitivity to the corresponding standard-of-care drugs (gemcitabine, sorafenib, and imatinib, respectively). An analysis of tumor cells derived from MIA PaCa-2 xenograft mice treated with h4#147D revealed that cell surface expression of CD147 and its binding partners, including CD44 and integrin α3β1/α6β1, was significantly reduced by h4#147D. Inhibition of focal adhesion kinase (FAK), activation of multiple stress responsible signal proteins such as c-JunN-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38MAPK), and expression of SMAD4, as well as activation of caspase-3 were obviously observed in the tumor cells, suggesting that h4#147D induced tumor shrinkage by inducing multiple stress responsible signals. These results suggest that the anti-CD147 antibody h4#147D offers promise as a new antibody drug candidate.

BRD4 degradation blocks expression of MYC and multiple forms of stem cell resistance in Ph+ chronic myeloid leukemia.

In most patients with chronic myeloid leukemia (CML) clonal cells can be kept under control by BCR::ABL1 tyrosine kinase inhibitors (TKI). However, overt resistance or intolerance against these TKI may occur. We identified the epigenetic reader BRD4 and its downstream‐effector MYC as growth regulators and therapeutic targets in CML cells. BRD4 and MYC were found to be expressed in primary CML cells, CD34+/CD38− leukemic stem cells (LSC), and in the CML cell lines KU812, K562, KCL22, and KCL22T315I. The BRD4‐targeting drug JQ1 was found to suppress proliferation in KU812 cells and primary leukemic cells in the majority of patients with chronic phase CML. In the blast phase of CML, JQ1 was less effective. However, the BRD4 degrader dBET6 was found to block proliferation and/or survival of primary CML cells in all patients tested, including blast phase CML and CML cells exhibiting the T315I variant of BCR::ABL1. Moreover, dBET6 was found to block MYC expression and to synergize with BCR::ABL1 TKI in inhibiting the proliferation in the JQ1‐resistant cell line K562. Furthermore, BRD4 degradation was found to overcome osteoblast‐induced TKI resistance of CML LSC in a co‐culture system and to block interferon‐gamma‐induced upregulation of the checkpoint antigen PD‐L1 in LSC. Finally, dBET6 was found to suppress the in vitro survival of CML LSC and their engraftment in NSG mice. Together, targeting of BRD4 and MYC through BET degradation sensitizes CML cells against BCR::ABL1 TKI and is a potent approach to overcome multiple forms of drug resistance in CML LSC.

CREB/Sp1-mediated MCL1 expression and NFκB-mediated ABCB1 expression modulate the cytotoxicity of daunorubicin in chronic myeloid leukemia cells

Although some studies have hinted at the therapeutic potential of daunorubicin (DNR) in chronic myeloid leukemia (CML), the mechanism by which DNR induces CML cell death is unclear. Therefore, this study aimed to investigate DNR-induced cell death signaling pathways in CML cell lines K562 and KU812. DNR-triggered apoptosis in K562 cells was characterized by inhibition of MCL1 expression, while restoration of MCL1 expression protected K562 cells from DNR-mediated cytotoxicity. In addition, DNR induced NOX4-dependent ROS production, leading to the activation of p38 MAPK and inactivation of Akt and ERK. Activated p38 MAPK stimulated protein phosphatase 2A-dependent dephosphorylation of CREB. Since Akt-mediated activation of ERK reduced β-TrCP mRNA stability, the inactivation of Akt-ERK axis increased β-TrCP expression, which in turn promoted proteasomal degradation of Sp1. Inhibition of CREB phosphorylation and Sp1 expression simultaneously reduced MCL1 transcription and protein expression. DNR-induced MCL1 suppression was not reliant on its ability to induce DNA damage. In addition, DNR induced the expression of drug exporter ABCB1 in K562 cells through the p38 MAPK/NFκB-mediated pathway, while imatinib or ABT-199 inhibited the DNR-induced effect. The combination of imatinib or ABT-199 with DNR showed synergistic cytotoxicity in K562 cells by increasing intracellular DNR retention. Cumulatively, our data indicate that DNR induces MCL1 downregulation in K562 cells by promoting p38 MAPK-mediated dephosphorylation of CREB and inhibiting the Akt-ERK axis-mediated Sp1 protein stabilization. Furthermore, experimental evidence indicates that DNR-induced death of KU812 cells occurs through a similar pathway.

Deciphering the Mechanisms of Osteoblast-Induced Resistance of Leukemic Stem Cell (LSC) in Ph+ CML: Role of PI3-Kinase, BRD4 and MYC and Development of Strategies to Overcome Osteoblast-Induced Resistance

Chronic myeloid leukemia (CML) is a stem cell neoplasms characterized by the chromosome translocation t(9;22) and the related BCR-ABL1 fusion gene. Therapy with BCR-ABL1 kinase inhibitors is highly effective in the treatment of CML and deep molecular responses are achieved in most patients. However, not all patients respond to these drugs due to resistance of leukemic stem cells (LSC). Recent data suggest that the disease-related microenvironment, known as the stem cell niche contributes to drug resistance and relapse in CML. So far, little is known about the resistance mechanisms protecting niche cells in the bone marrow of patients with CML. We have recently shown that osteoblasts are a major site of LSC-mediated resistance against BCR-ABL1-targeting drugs in CML. In the current study, we screened for drugs that are able to suppress growth and viability of osteoblasts and/or other niche-related cells and can thereby overcome drug resistance of CML LSC. Proliferation was analyzed by determining 3H-thymidine uptake in niche-related cells and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC 50: 0.05 µM; copanlisib IC 50: 0.05 µM), the osteoblastic cell line CAL-72 (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 1 µM), primary human umbilical vein endothelial cells (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 0.5 µM) and the endothelial cell line HMEC-1 (BEZ235 IC 50: 1 µM; copanlisib IC 50: 1 µM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. As determined by flow cytometry, BEZ235 and copanlisib suppressed the expression of phosphorylated (p) pAKT and pS6 in endothelial cells and osteoblasts whereas rapamycin downregulated pS6 expression but did not decrease expression of pAKT. Moreover, we found that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing growth and viability of osteoblasts and endothelial cells. Furthermore, BEZ235 and copanlisib were found to overcome osteoblast-induced resistance of K562, KU812 cells, and primary CD34 +/CD38 − CML LSC against nilotinib and ponatinib. This effect was also seen when CAL-72 cells were first exposed to BEZ235 or copanlisib and washed before co-cultures with CML cells and BCR-ABL1 inhibitors were prepared, suggesting that osteoblast inhibition was a crucial event capable of disrupting LSC resistance in these co-cultures. Of all other drugs tested, only the BRD4-targeting drug JQ1 was found to suppress CAL72-induced resistance in the CML cell lines KU812 and K562, suggesting that osteoblast-induced resistance of CML cells is also mediated by a BRD4-MYC pathway. In a next step, we examined the expression of resistance-mediating immune checkpoint molecules on CML cells (KU812, K562, LSC) and on osteoblasts by flow cytometry. We found that CML cells and CAL72 cells constitutively express PD-L1 and that interferon-gamma (IFN-G) promotes the expression of PD-L1 in all cell types tested. Moreover, we found that the BRD4 blocker JQ1 and the BRD4-degrader dBET6 suppress the IFN-G-induced upregulation of PD-L1 in CML LSC and osteoblasts. In conclusion, our data show that osteoblast-induced resistance of CML stem cells is mediated by a PI3K-dependent pathway and BRD4/MYC, and that BRD4-inhibition or BRD4-degradation counteracts osteoblast-induced resistance of CML (stem) cells against BCR-ABL1 inhibitors and PD-L1 expression on CML LSC and osteoblasts. We hypothesize that checkpoint inhibition may assist in drug-induced eradication of CML LSC and thus in the development of curative drug therapies in Ph + CML. DisclosuresHoermann: Novartis: Honoraria. Gleixner: Pfizer: Honoraria; Abbvie: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Sperr: AbbVie, BMS-Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Novartis, Pfizer, StemLine, Thermo Fisher: Honoraria, Research Funding. Valent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

Secondary basophilic leukemia in Ph-negative myeloid neoplasms: A distinct subset with poor prognosis.

During progression of myeloid neoplasms, the basophil compartment may expand substantially and in some of these patients, a basophilic leukemia is diagnosed. In patients with Ph-chromosome+ chronic myeloid leukemia, acceleration of disease is typically accompanied by marked basophilia. In other myeloid neoplasms, secondary leukemic expansion of basophils is rarely seen. We report on 5 patients who suffered from a myelodysplastic syndrome, myeloproliferative neoplasm, or acute leukemia and developed a massive expansion of basophils during disease progression. In 4 of 5 patients, peripheral blood basophil counts reached 40%, and the diagnosis “secondary basophilic leukemia” was established. As assessed by flow cytometry, neoplastic basophils expressed CD9, CD18, CD25, CD33, CD63, PD-L1, CD123, and CLL-1. In addition, basophils were found to display BB1 (basogranulin), 2D7, tryptase and KIT. In 4 of 5 patients the disease progressed quickly and treatment with azacitidine was started. However, azacitidine did not induce major clinical responses, and all patients died from progressive disease within 3 Y. In in vitro experiments, the patients´ cells and the basophilic leukemia cell line KU812 showed variable responses to targeted drugs, including azacitidine, venetoclax, hydroxyurea, and cytarabine. A combination of venetoclax and azacitidine induced cooperative antineoplastic effects in these cells. Together, secondary basophilic leukemia has a poor prognosis and monotherapy with azacitidine is not sufficient to keep the disease under control for longer time-periods. Whether drug combination, such as venetoclax+azacitidine, can induce better outcomes in these patients remains to be determined in future clinical studies.

AKR1C3 decreased CML sensitivity to Imatinib in bone marrow microenvironment via dysregulation of miR-379-5p

BackgroundDrug resistance is an important cause of death for most patients with chronic myeloid leukemia (CML). The bone marrow microenvironment is believed to be mainly responsible for resistance to BCR-ABL tyrosine kinase inhibitors. The mechanism involved, however, is still unclear. MethodsBioinformatic analysis from GEO database of AKR1C3 was utilized to identify the AKR1C3 expression in CML cells under bone marrow microenvironment. Western blot and qPCR were performed to detect the AKR1C3 expression in two CML cell lines K562 and KU812 cultured +/‐ bone microenvironment derived stromal cells. CCK-8, soft agar colony assay, and Annexin V/PI assay were performed to detect the sensitivity of CML cells (K562 and KU812) to Imatinib under a gain of or loss of function of AKR1C3 treatment. The CML murine model intravenous inoculated with K562-OE-vector and K562-OE-AKR1C3 cells were established to estimate the effect of AKR1C3 inhibitor Indomethacin on Imatinib resistance. The bioinformatic analysis of miRNA databases was used to predict the potential miRNAs targeting AKR1C3. And the luciferase assay was utilized to validate the target relationship between miR-379-5p and AKR1C3. And, the soft agar colony assay and Annexin V/PI were used to validate the effect of miR-379-5p in AKR1C3 induced Imatinib resistance. ResultsIn present study, we investigated AKR1C3 was highly expressed in CML under bone marrow microenvironment. AKR1C3 decreased Imatinib activity in K562 and KU812 cells, while inhibition of AKR1C3 could enhance Imatinib sensitivity in vitro study. Furthermore, murine model results showed combination use of AKR1C3 inhibitor Indomethacin effectively prolong mice survival, indicating that AKR1C3 is a promising target to enhance Imatinib treatment. Mechanically, AKR1C3 was found to be suppressed by miR-379-5p, which was down-expression in bone marrow microenvironment. Besides, we found miR-379-5p could bind AKR1C3 3’UTR but not degrade its mRNA level. Further, gain of miR-379-5p rescued the imatinib resistance induced by AKR1C3 overexpression in CML cells. ConclusionsAltogether, our study identifies a novel signaling regulation of miR-379-5p/AKR1C3/EKR axis in regulating IM resistance in CML cell, and provides a scientific base for exploring AKR1C3 as a biomarker in impeding IM resistance in CML.

BRD4 Degradation Is a Potent Approach to Block MYC Expression and to Overcome Multiple Forms of Stem Cell Resistance in Ph+ CML

Chronic myeloid leukemia (CML) is a hematopoietic stem cell neoplasm in which BCR-ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients, leukemic cells can be kept under control by BCR-ABL1 tyrosine kinase inhibitors (TKI). Nevertheless, resistance against one or more TKI may occur. Therefore, research is focusing on novel potential drug targets in CML. We have recently identified the epigenetic reader bromodomain-containing protein 4 (BRD4) as a new therapeutic target in leukemic stem cells (LSC) in acute myeloid leukemia. In the present study, we examine the expression of BRD4 and its downstream effector MYC in CML cells and asked whether BRD4 serves as a drug target in CML cells and whether BRD4-targeting drugs, including JQ1 and newly developed BRD4 degraders (dBET1 and dBET6) are able to overcome LSC resistance in CML. Primary CML cells were obtained from 22 patients with chronic phase (CP) CML and 3 with blast phase (BP) CML. As determined by qPCR and/or immunocytochemistry, the CML cell lines KU812 and K562 as well as primary CML cells expressed BRD4 and MYC. All three BRD4-targeting drugs (JQ1, dBET1 and dBET6) were found to decrease MYC expression in KU812 and K562 cells as assessed by Western blotting. In 3H-thymidine uptake experiments, JQ1 and dBET6 were found to inhibit the proliferation of KU812 in a dose-dependent manner (IC50, JQ1: 100-500 nM; dBET6: 50-100 nM) whereas dBET1 showed only little if any effects on growth of KU812 cells (IC50: 1-5 µM), and in K562 cells, only dBET6 was found to inhibit growth with a reasonable IC50 value (250-500 nM). Corresponding results were obtained when examining drug effects on survival of CML cell lines by Annexin-V/PI staining. All three BRD4-targeting drugs were found to inhibit proliferation of primary CP CML cells with varying IC50 values. As expected, growth-inhibitory effects of dBET6 were more pronounced (IC50: <100 nM) compared to effects seen with JQ1 and dBET1. dBET1 and dBET6 were also found to inhibit growth of primary CML cells obtained from patients with BP CML, whereas JQ1 was not effective. JQ1 also failed to suppress survival on CML CD34+/CD38− LSC. By contrast, dBET1 induced apoptosis in CML LSC at 1 µM and dBET6 induced apoptosis in CML LSC at 0.1 µM. dBET6 induced apoptosis in CML LSC obtained from patients with imatinib-sensitive CML as well as patients with imatinib-resistant CML harboring BCR-ABL1 T315I or BCR-ABL1 F317L. Finally, pre-incubation of CD34+ CP CML cells with dBET6 resulted in reduced leukemic engraftment in NSG mice exhibiting human membrane-bound stem cell factor, SCF [NSG-Tg(hu-mSCF)] 6 months after transplantation (engraftment with CD45+/CD33+/CD19−cells in control mice receiving DMSO-treated cells: 8.1±6.6% vs mice receiving dBET6-treated cells: 1.1±0.6%). To further explore the ability of dBET6 to interfere with LSC resistance in CML, we established a co-culture system mimicking LSC-niche interactions in the osteoblastic niche. In this model, co-culturing K562 cells, KU812 cells or primary CML LSC with the osteoblast-like osteosarcoma cell line CAL-72 resulted in resistance against nilotinib and ponatinib. In this culture system, JQ1 was found to partially restore TKI effects in K562 cells and completely restored TKI effects in KU812 cells. Interestingly, JQ1 was not able to restore TKI effects in primary CML LSC in these co-cultures. However, dBET6 was found to overcome niche cell-induced TKI-resistance of primary CML LSC. Finally, we were able to demonstrate that JQ1, dBET1 and dBET6 inhibit interferon-gamma-induced upregulation of PD-L1 expression in CML LSC. Together we show that BRD4 and MYC are potential new therapeutic drug targets in CML and that the BET-degrader dBET6 overcomes multiple forms of LSC resistance, including i) intrinsic resistance, ii) mutation-induced resistance, iii) niche induced resistance and iv) checkpoint-mediated resistance. Whether BRD4 degradation is also able to overcome TKI-resistance of BCR-ABL1+ LSC in vivo in patients with CML remains to be determined in clinical trials. DisclosuresHoermann:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria. Wolf:BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding. Mayer:Amgen: Research Funding; Novartis: Research Funding. Zuber:Mirimus Inc.: Consultancy, Other: Shareholder; Boehringer Ingelheim GmbH & Co KG: Research Funding. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Valent:Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria.

Abstract 3739: Nuclear hormone receptors modulate imatinib resistance in chronic myeloid leukemia (CML)

Abstract Despite the impressive success of molecular targeted therapy in chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKI) such as imatinib (IM), a proportion of patients exhibit suboptimal response and intolerance to the drug. While the mechanism by which resistance or intolerance develops is not completely clear, it is possible that several secondary modifiers could determine disease progression and drug response in CML. Nuclear Hormone Receptors-NHRs are attractive but less explored therapeutic targets in myeloid leukemia. The impressive success of treatment with ATRA, a RAR ligand, in the treatment of APL has not been translated to other myeloid leukemia subtypes. Recently, Prost et al., 2015 showed that Peroxisome proliferator-activated receptor-γ agonist Pioglitazone eliminates the residual CML stem cell pool and these patients remained in complete molecular response even after withdrawal of IM. The role of other NHRs in CML TKI resistance has not been explored extensively. Based on the in-vitro drug sensitivity data, CML cell lines KU812, EM2 were grouped as sensitive (IC50-0.15 &amp; 2.29uM) and Lama84, KCL22 were grouped as resistant (IC50-54.45 &amp; 66uM) to IM. The NHRs and coregulators that are differentially expressed between IM sensitive and resistant cell lines were identified using NHR RT2 PCR profiler array (SAbiosciences). Based on +2 fold cutoff, the differentially expressed NHRs between test and control group were identified. Eight NHRs (AHR, AR, ESR1, ESRRG, PPARγ, RXRA, RXRB &amp; THRA) were upregulated in the sensitive compared to the resistant cell lines, which was further validated by qRT-PCR. To understand the effect of these NHRs in sensitizing resistant CML cells to Imatinib, KCL22 and Lama84 cells were pretreated with NHR ligands, followed by increasing concentrations of Imatinib (5µM-100µM). Ligands of RXR (9-cis retinoic acid), AR (testosterone) and ESRRG1 (GSK4716) significantly improved IM sensitivity by increasing the RNA expression of IM influx transporter hOCT1, which was also reported by Wang et al., 2012; Austin et al., 2014 using ligands for RXR, PXR and PPARγ. Pretreatment with these ligands did not have significant effect on the mRNA expression of BCR-ABL, but there was increased inhibition of the BCR-ABL downstream targets such as pCRKL, pSTAT5 and pAKT at the protein level in combination with IM. The AR, RXR &amp; ESRRG ligands significantly decreased the IC50 to IM (p&amp;lt;0.0001) in primary CML cells (n=22) in combination with IM. We also observed significant sensitization of primary samples (n=7) to IM when used in combination with clinically available RXR and AR ligands isotretinoin (p=0.0011) &amp; stanozolol (p=0.0054). Our finding suggests that NHR ligands, especially RXR, AR &amp; ESRRG when used in combination with IM could overcome resistance. Further studies are warranted to confirm the mechanistic effects of these ligands in modulating drug resistance in CML. Citation Format: Bharathi M. Rajamani, Sreeja Karathedath, Raveen Stephen Illangeswaran, Esther Sathya Bama Benjamin, Vikram Mathews, Aby Abraham, Poonkuzhali Balasubramanian. Nuclear hormone receptors modulate imatinib resistance in chronic myeloid leukemia (CML) [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 3739.

Structure-based design of novel quinoxaline-2-carboxylic acids and analogues as Pim-1 inhibitors

We identified a new series of quinoxaline-2-carboxylic acid derivatives, targeting the human proviral integration site for Moloney murine leukemia virus-1 (HsPim-1) kinase. Seventeen analogues were synthesized providing useful insight into structure-activity relationships studied. Docking studies realized in the ATP pocket of HsPim-1 are consistent with an unclassical binding mode of these inhibitors. The lead compound 1 was able to block HsPim-1 enzymatic activity at nanomolar concentrations (IC50 of 74 nM), with a good selectivity profile against a panel of mammalian protein kinases. In vitro studies on the human chronic myeloid leukemia cell line KU812 showed an antitumor activity at micromolar concentrations. As a result, compound 1 represents a promising lead for the design of novel anticancer targeted therapies.

Epithelial cell-basophil interactions stimulate the production of IL-5 in innate allergic inflammation

Epithelial cell-derived interleukin (IL)-33 plays an important role in the initiation and activation of innate allergic inflammation by stimulating the production of Th2 cytokines. It act as a cytokine through the transmembrane receptor ST2L, which is expressed in most of the inflammatory cells, and its activity is neutralized by the soluble spliced variant of ST2 (sST2). Basophil expresses ST2L and produces IL-5 in response to IL-33, however, the role of epithelial cell-basophil interactions in allergic inflammation is still unclear. Cultured Normal Human bronchial epithelial (NHBE) cells and human basophil cell line KU812 cells were used to study the epithelial cell-basophil interactions in the house dust mite (HDM)-induced production of IL-5. Production of IL-5, IL-33 and sST2 was evaluated by ELISA. HDM stimulated the rapid release of IL-33 from cultured NHBE cells at 1 hour after the incubation. Basophil cell line KU812 cells produced IL-5 in response to IL-33, and the conditioned medium of HDM-stimulated NHBE cells stimulated IL-5 production from KU812 cells. Co-culture of NHBE cells with KU812 cells stimulated the production of sST2 time-dependently in response to HDM, and HDM did not stimulate the production of IL-5 in co-cultured cells. These results indicate that epithelial cell-derived IL-33 stimulates the production of IL-5 from basophils, and sST2 is an important regulator of IL-33 in innate allergic inflammation.

BTK inhibition is a potent approach to block IgE-mediated histamine release in human basophils.

BackgroundRecent data suggest that Bruton's tyrosine kinase (BTK) is an emerging therapeutic target in IgE receptor (IgER)‐cross‐linked basophils.MethodsWe examined the effects of four BTK inhibitors (ibrutinib, dasatinib, AVL‐292, and CNX‐774) on IgE‐dependent activation and histamine release in blood basophils obtained from allergic patients (n=11) and nonallergic donors (n=5). In addition, we examined the effects of these drugs on the growth of the human basophil cell line KU812 and the human mast cell line HMC‐1.ResultsAll four BTK blockers were found to inhibit anti‐IgE‐induced histamine release from basophils in nonallergic subjects and allergen‐induced histamine liberation from basophils in allergic donors. Drug effects on allergen‐induced histamine release were dose dependent, with IC 50 values ranging between 0.001 and 0.5 μmol/L, and the following rank order of potency: ibrutinib>AVL‐292>dasatinib>CNX‐774. The basophil‐targeting effect of ibrutinib was confirmed by demonstrating that IgE‐dependent histamine release in ex vivo blood basophils is largely suppressed in a leukemia patient treated with ibrutinib. Dasatinib and ibrutinib were also found to counteract anti‐IgE‐induced and allergen‐induced upregulation of CD13, CD63, CD164, and CD203c on basophils, whereas AVL‐292 and CNX‐774 showed no significant effects. Whereas dasatinib and CNX‐774 were found to inhibit the growth of HMC‐1 cells and KU812 cells, no substantial effects were seen with ibrutinib or AVL‐292.Conclusions BTK‐targeting drugs are potent inhibitors of IgE‐dependent histamine release in human basophils. The clinical value of BTK inhibition in the context of allergic diseases remains to be determined.

Identification of CD25 as STAT5-Dependent Growth Regulator of Leukemic Stem Cells in Ph+ CML.

In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs. CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25(+)CML cell line KU812. In contrast to normal hematopoietic stem cells, CD34(+)/CD38(-)CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin(-)/Sca-1(+)/Kit(+)stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cellsin vitroand their engraftmentin vivoin NOD/SCID-IL-2Rγ(-/-)mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib. CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML.

Discovery of 2-((3-Amino-4-methylphenyl)amino)-N-(2-methyl-5-(3-(trifluoromethyl)benzamido)phenyl)-4-(methylamino)pyrimidine-5-carboxamide (CHMFL-ABL-053) as a Potent, Selective, and Orally Available BCR-ABL/SRC/p38 Kinase Inhibitor for Chronic Myeloid Leukemia.

Starting from a dihydropyrimidopyrimidine core scaffold based compound 27 (GNF-7), we discovered a highly potent (ABL1: IC50 of 70 nM) and selective (S score (1) = 0.02) BCR-ABL inhibitor 18a (CHMFL-ABL-053). Compound 18a did not exhibit apparent inhibitory activity against c-KIT kinase, which is the common target of currently clinically used BCR-ABL inhibitors. Through significant suppression of the BCR-ABL autophosphorylation (EC50 about 100 nM) and downstream mediators such as STAT5, Crkl, and ERK's phosphorylation, 18a inhibited the proliferation of CML cell lines K562 (GI50 = 14 nM), KU812 (GI50 = 25 nM), and MEG-01 (GI50 = 16 nM). A pharmacokinetic study revealed that 18a had over 4 h of half-life and 24% bioavailability in rats. A 50 mg/kg/day dosage treatment could almost completely suppress tumor progression in the K562 cells inoculated xenograft mouse model. As a potential useful drug candidate for CML, 18a is under extensive preclinical safety evaluation now.

Modulation of Leukotriene Signaling Affecting Cell Growth in Chronic Myeloid Leukemia: A Key Pathway to Cure?

▪Background: Recent data suggest that tyrosine kinase inhibitor (TKI)-insensitive leukemic stem cells often prevail in chronic myeloid leukemia (CML) patients subjected to long-term TKI treatment. To achieve cure additional aberrant pathways in these patients, such as leukotriene (LT) signaling, may need to be targeted. We have previously shown that human CML cells have an increased capacity to produce LT and that LT can stimulate normal myelopoiesis (Tornhamre ExpHem 2003). Chen later noted a striking up-regulation of ALOX5/5LO (catalyzing an initial step in LT formation) in CML mice, and that inhibition of this enzyme improved survival of these animals by a magnitude similar to that induced by imatinib (Chen NatureGen 2009). Here wexve assessed the effect of LT modulating agents on the growth of human CML cells in vitro.Materials and methods: Human CML cells, derived from the three cell lines K562, Kcl22 and KU812, were grown in microtitre plate cultures, in the presence of RPMI 1640. The MTT technique was used to evaluate the number of viable cells at 72 hours. Protein expression was assessed by Western blot and immunocytochemistry.Results: Several specific modulators of LT-signaling were capable of inducing dose-dependent growth inhibitory effects on CML cells. Thus, the cysteinyl-LT receptor antagonist montelukast, a drug with approved clinical use in human asthma, was shown to reduce the growth of all three tested CML cell lines. In Fig. 1 this is examplified by K562 cells, where also additive effects between montelukast and imatinib are indicated. All cell lines expressed the cysLT1-receptor. Furthermore, the LTB4 receptor antagonist etalocib, as well as the 5-LO activating protein (FLAP) inhibitors licofelone and Bay-X-1005, also executed inhibitory effects at concentrations considered as physiological. As expected, addition of the TKIs imatinib, nilotinib or dasatinib to the cultures also generated dose-dependent inhibitory effects on the growth of all tested CML cell lines. An LD50 of approximately 0.5 µM, 10 nM and 0.1 nM was noted for imatinib, nilotinib and dasatinib, respectively, for K562 cells at 72 hours.Conclusions: We demonstrate that several compounds, known to specifically inhibit leukotriene signaling by different mechanisms, were at clinically relevant concentrations capable of significantly suppressing the growth of human CML cells. Some of these compounds are already in clinical practice for non-hematological disorders. They could provide an additional therapeutic possibility in the quest to cure CML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of the Epigenetic Reader BRD4 As a Novel Potential Target in Ph+ CML

Chronic myelogenous leukemia (CML) is a bone marrow-derived hematopoietic neoplasm in which BCR/ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients with CML, leukemic cells can be kept under control by BCR/ABL1 tyrosine kinase inhibitors (TKI), including imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. Nevertheless, resistance or intolerance against one or more of these TKI may occur. Therefore, current research is focusing on novel potential drug targets in CML. A promising class of targets may be epigenetic regulators of cell growth, such as members of the bromodomain and extra-terminal domain (BET) family. The epigenetic reader and BET family member BRD4 has recently been identified as a novel potential drug target in acute myeloid leukemia (AML). However, so far, little is known about the expression and function of BRD4 in CML cells. The aims of the present study were to determine the expression of BRD4 and its downstream target MYC in CML cells and to explore whether BRD4 can serve as a novel drug target in this disease. As determined by qPCR, primary CML cells (chronic phase patients, n=7) as well as the CML cell lines KU812 and K562 expressed BRD4 mRNA. In addition, both CML cell lines stained positive for BRD4 in our immunocytochemistry staining experiments. In one patient with accelerated phase CML, putative leukemic (CD34+/CD38-) stem cells were sorted to near homogeneity and found to express BRD4 mRNA by qPCR. In order to examine the functional role of BRD4 in CML cells, a BRD4-specific shRNA was applied. In these experiments, the shRNA-induced knockdown of BRD4 in KU812 cells and K562 resulted in reduced growth compared to a control shRNA. Furthermore, the BRD4-targeting drug JQ1 was found to inhibit 3H-thymidine uptake and thus proliferation in KU812 cells in a dose-dependent manner (IC50: 0.25-0.75 µM). In addition, we were able to show that JQ1 inhibits growth of primary CML cells with variable IC50 values (0.1-5 µM). However, no substantial growth-inhibitory effects of JQ1 were seen in K562 cells (IC50: >5 µM). As determined by Annexin V/PI staining, JQ1 induced apoptosis in KU812 cells whereas no apoptosis-inducing effect of JQ1 was observed in K562 cells. Nevertheless, we were able to show that both CML cell lines as well as primary CML cells express MYC mRNA, and treatment of KU812 cells or K562 cells with JQ1 resulted in a decreased expression of MYC mRNA and MYC protein. Next, we analyzed whether MYC expression in CML cells can be blocked by BCR/ABL1 TKI. We found that imatinib, nilotinib, dasatinib, and ponatinib decrease MYC mRNA- and MYC protein expression in KU812 and K562 cells. Finally, we found that JQ1 cooperates with imatinib, nilotinib, ponatinib and dasatinib in inhibiting the proliferation of KU812 and K562 cells. Together, our data show that BRD4 serves as a potential new target in CML cells, and that the BRD4 blocker JQ1 cooperates with BCR/ABL1 TKI in inducing growth-inhibition. Whether BRD4 inhibition is a pharmacologically meaningful approach in patients with TKI-resistant CML remains to be determined in clinical trials. DisclosuresSperr:Ariad: Consultancy; Celgene: Consultancy. Zuber:Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.