The HACE1-NRF2 Axis a Novel Target in Acute Myeloid Leukemia

Abstract Acute Myeloid Leukemia (AML) is a genetically heterogenous disease characterized by clonal expansion of immature myeloid progenitors cells in the bone marrow (BM). Despite this genetic heterogeneity, AML patients share Leukemia associated oncogenes such as NF-E2-related factor 2 (Nrf2) (Rushworth SA et al.). NRF2 is a transcription factor that activates genes with antioxidant response elements (ARE)-containing promoters and protects cancer cells from apoptosis. Inhibition of NRF2 or antioxidant defense increases the level of Radical Oxygen Species (ROS), leading to tumor supression (Chio IIC et al.). Recently, the E3 Ubiquitin-Protein Ligase HACE1, a tumor suppressor in solid tumors, was demonstrated to promote the expression of NRF2 in Huntigton disease (Rotblat B et al.). Thus, we hypothesized a role for HACE1 as an oncogenic factor acting through NRF2 activation in myeloid malignancies and provide first data supporting the HACE1-NRF2 axis to be a novel target in acute myeloid leukemias. Material and methods The mRNA expression data from AML patients (296 samples) vs normal Hematopoietic Stem Cells (HSC) (6 samples) were exported from the bloodSpot database. HACE1 mRNA and protein expression was measured by q-RT-PCR and western blot in 12 commercially available Myeloid Malignancies cell lines. The HACE1 inducible knock down (KD) was carried out by Sleeping Beauty Transposon system in U937 and NOMO-1 cell lines. The cell viability was analyzed by Cell Titer Glo Luminescent assay. Apoptosis was measured by Annexin V (AV)/Propidium Iodide (PI) assay. Results and discussion HACE1 mRNA is downregulated in AML patients compared to HSC (***p<0.001, Bloodspot database). However mRNA and HACE1 protein expression do not correlate in AML cell lines, suggesting post translational modifications. High HACE1 protein expression was observed in most AML cell lines. HACE1 KD reduced drastically the cell viability of U937 cells through caspase activation and NRF2 degradation. However, no effect on cell viability was observed in NOMO-1 cells. Recently, non-programmed cell death necroptosis induction has been described by TNFR1 activation in HACE1 knock out Mouse Embrionic Fibroblast cells (Tortola L et al.). In line with this study, we observed that TNF induces strong cell death in HACE1 KD NOMO-1 cells within 48 hours. In addition HACE1 KD promotes autophagy through p62 degradation (late autophagy marker) in U937 cells. Autophagy has recently been described to contribute to the differentiation and death of AML cells, and to the promotion of immunostimulatory signals activating immune responses against cancer cells (Chen L et al.; Pietrocola F et al.). Thus HACE1 might be a potential target to induce autophagy, providing a novel therapeutical target in the treatment of myeloid malignancies. Finally, HACE1 KD in our hands promoted sensitization of U937 and NOMO-1 cells to cytatarabine, the backbone therapy in AML patients. This treatment promotes HACE1 protein expression at 24 and 48 hours in NOMO-1 cells, which may explain the better response rates of HACE1 KD cells to cytarabine. Taken together, we provide first evidence of HACE1 being a novel oncogene in AML and that the HACE1-NRF2 axis is a promising target in the treatment of Acute Myeloid Leukemias. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Inhibition of IRAK1 As a Novel Therapeutic Strategy in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v118.21.612.612 ◽

2011 ◽

Vol 118 (21) ◽

pp. 612-612 ◽

Cited By ~ 1

Author(s):

Garrett Rhyasen ◽

Lyndsey Bolanos ◽

Jing Fang ◽

Carmen Rigolino ◽

Agostino Cortelezzi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Expression ◽

Myelodysplastic Syndrome ◽

Cell Lines ◽

Myeloid Leukemia ◽

Kinase Activity ◽

Myeloid Malignancies ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Abstract 612 Recent work has shown that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients exhibit downregulation of miR-146a, a miRNA that negatively regulates the innate immune pathway by targeting IRAK1 and TRAF6. Mice lacking miR-146a show elevated IRAK1 protein expression, and develop AML and MDS-like features resembling the human diseases. Prior to this study, the role of IRAK1 in human myeloid malignancies was unknown. We conducted a comparison of gene expression profiles of 136 cases of MDS CD34+ cells with 17 normal CD34+ cells obtained from ArrayExpress (E-GEOD-19429; Pellagatti et al., Leukemia, 2010). According to this data set, we observed IRAK1 overexpression in MDS patients (P = 0.017). IRAK1 is a serine/threonine kinase, and after phosphorylation on threonine-209 (T209), its kinase activity is induced, thus allowing for subsequent activation of TRAF6 and eventually NF-kB. Interestingly, we observed higher basal levels of phospho-IRAK1 at T209 in MDS and AML samples as compared to normal human CD34+ cells. To investigate the potential role of IRAK1 in AML and MDS, we used genetic and pharmacological approaches to suppress IRAK1 activity in MDS/AML cell lines and bone marrow cells from MDS patients. RNAi-mediated knockdown of IRAK1 in MDS and AML samples resulted in impaired growth of malignant hematopoietic stem/progenitor cells in methylcellulose assays and rapid apoptosis in vitro. In addition, we used a small-molecule inhibitor (benzimidazole analog; Amgen Inc.) to potently inhibit IRAK1 kinase activity. MDS/AML cell lines and MDS patient samples cultured with the IRAK1 inhibitor exhibited impaired growth and increased apoptosis, which coincided with decreased phospho-IRAK1 at T209, and active versions of TRAF6 and NF-kB. Importantly, the inhibition of IRAK1 kinase function is selectively detrimental to MDS and AML samples while preserving normal CD34+ cell viability and function. Given this novel requirement of IRAK1 in MDS and AML, we examined whether Lenalidomide or Bortezomib, two treatment options for MDS/AML and reported immunosuppressors, exhibit anti-leukemic activity in part by targeting IRAK1. We observed that Bortezomib, but not Lenalidomide, inhibits IRAK1 mRNA and protein expression in MDS/AML cells. The cytotoxic effect of Bortezomib can be partly rescued by forced expression of IRAK1 in these cells. To determine the molecular and cellular basis of cell death following loss of IRAK1 function or expression, we applied microarrays to MDS cells treated with IRAK1 inhibitor or transduced with a lentiviral vector encoding an shRNA targeting IRAK1. An overlap of commonly deregulated genes imposed by loss of IRAK1 expression or by the IRAK1 inhibitor revealed unique pathways relevant to the survival of MDS and AML cells. In summary, these findings are the first to implicate IRAK1 in the maintenance of myeloid malignancies and describe the effectiveness of an IRAK1 inhibitor on suppressing MDS and AML viability. Disclosures: Oliva: Celgene: Consultancy.

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Novel Therapy for FLT3-ITD Acute Myeloid Leukemia Utilizing the Combination of CUDC-907 and Gilteritinib

Blood ◽

10.1182/blood-2018-99-111177 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1427-1427 ◽

Cited By ~ 3

Author(s):

Tristan Knight ◽

Xinan Qiao ◽

Holly Edwards ◽

Hai Lin ◽

Jeffrey W. Taub ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Expression ◽

Tyrosine Kinase ◽

Cell Lines ◽

Receptor Tyrosine Kinase ◽

Myeloid Leukemia ◽

Dual Inhibitor ◽

Western Blots ◽

Flt3 Expression ◽

Acute Myeloid

Abstract Introduction: FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase, and is mutated in approximately one third of acute myeloid leukemia (AML) patients; this mutation confers a poor prognosis. Two FLT3 mutations are commonly seen in AML: internal tandem duplications (ITD) in the juxtamembrane domain (~25% of AML), and point mutations in the receptor tyrosine kinase at codon 835 (D835) (~7% of AML). Both mutations result in constitutive FLT3 activation, causing downstream activation of multiple pathways, in particular, those involved in cell survival including the RAS-RAF-MEK-ERK, JAK-STAT5, and PI3K/AKT pathways. PI3K-AKT may also be activated by AXL, also a tyrosine kinase, via its targets PLC, Grb2, and PI3K. Logically, then, inhibition of FLT3 is a promising pharmacological approach for treating this subtype of AML. Gilteritinib (ASP-2215) is a novel dual inhibitor of FLT3 and AXL, exposure to which results in upregulation of FLT3 as a resistance mechanism. Previously, we found that the novel dual PI3K/histone deacetylase (HDAC) inhibitor CUDC-907 downregulates FLT3 expression in AML cells (Figure 1A). Additionally, inhibition of FLT3 and AXL by gilteritinib may not result in robust inactivation of both the PI3K-Akt and MEK/ERK pathways due to crosstalk between the two pathways. Thus, our hypothesis was that CUDC-907 would sensitize AML cells to gilteritinib, resulting in concurrent inhibition of all the downstream signaling pathways of FLT3 and AXL, leading to synergistic antileukemic activities again FLT3-mutated AML (Figure 1B). Methods: FLT3-ITD AML cell lines (MV4-11 and MOLM-13) and primary patient samples were treated with CUDC-907, gilteritinib, both, or neither for 24 hours, at clinically achievable concentrations. Annexin V/Propidium Iodide (PI) staining and flow cytometry analyses was performed, and combination indexes (CI) calculated; CI<1, CI=1, and CI>1 indicating synergistic, additive, or antagonistic effects, respectively. Western blots were performed after treatment for 0-24 hours to determine protein expression of relevant targets. Results: CUDC-907 and gilteritinib demonstrated potent synergistic antileukemic effects in FLT3-ITD AML cell lines and FLT3-ITD patient samples (AML#171, AML#180), the combination exceeding either in isolation (Figure 1C). These findings were confirmed via western blot, which showed accentuated upregulation of cleaved caspase3 with combination therapy, in both cell lines and one patient sample, demonstrating drug-induced apoptosis. We confirmed that CUDC-907 abolishes gilteritinib-induced expression of FLT3 in a time-dependent fashion in cell lines MV4-11 and MOLM-13 (Figure 1D). Gilteritinib treatment decreased p-AKT, p-S6, and p-STAT5, while inhibition of the ERK pathway, as assessed by p-ERK expression, varied amongst the samples (Figure 1E). CUDC-907 treatment decreased both p-AKT and p-ERK. MOLM-13 cells showed increased p-ERK following gilteritinib treatment and increased p-STAT5 after CUDC-907 treatment. In all samples, combination of gilteritinib with CUDC-907 resulted in decrease of p-STAT5 and p-S6, similar to gilteritinib treatment alone, and further reduction of p-AKT and p-ERK compared to single drug treatments. Gilteritinib treatment also reduced expression of anti-apoptotic protein Mcl-1, which was further decreased in combination treated cells. Subsequently, time-course analysis was performed in both cell lines; findings were consistent with prior observations, and confirmed that protein expression changed over time, in relation to gilteritinib/CUDC-907/combined treatment exposure. Conclusion: We confirmed that CUDC-907 and Gilteritinib synergistically induce apoptosis in both cell lines and primary patient samples derived from patients with FLT3-ITD AML, and that CUDC-907 abolishes Gilteritinib-induced FLT3 expression. Additionally, the combination cooperatively inhibits the PI3K-AKT, JAK-STAT, and RAS-RAF pathways, while preventing escape via alternative pathways. Our results provide a strong foundation for subsequent in vivo murine studies, and eventual clinical evaluation of the combination of gilteritinib and CUDC-907 for the treatment of AML. Figure 1. Figure 1. Disclosures Ge: MEI Pharma: Research Funding.

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Degradation of TRAF6 by Bortezomib-Induced Autophagy Contributes to Cell Death in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v118.21.2452.2452 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2452-2452

Author(s):

Jing Fang ◽

Lyndsey Bolanos ◽

Garrett Rhyasen ◽

Carmen Rigolino ◽

Agostino Cortelezzi ◽

...

Keyword(s):

Cell Death ◽

Acute Myeloid Leukemia ◽

Myelodysplastic Syndrome ◽

Cell Lines ◽

Myeloid Leukemia ◽

26S Proteasome ◽

Negative Regulator ◽

Myeloid Malignancies ◽

Chromosome 5Q ◽

Acute Myeloid

Abstract Abstract 2452 Deletion of chromosome 5q in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients results in loss of miR-146a, which is a negative regulator of the innate immune pathway by targeting TNF receptor associated factor-6 (TRAF6). Therefore, MDS and AML patients with reduced miR-146a expression concomitantly exhibit elevated TRAF6 protein. TRAF6 is an E3 ubiquitin ligase that catalyzes K63-linked polyubiquitin chains on substrates that lead to pathway activation, one of which includes NF-kB. Mice lacking miR-146a, or with overexpression of TRAF6, develop AML- and MDS-like features. Bortezomib (Velcade©), which shows promise alone or in combination with chemotherapy in certain groups of MDS and AML patients, is a selective and reversible inhibitor of the 26S proteasome. Studies on the mechanism of action of Bortezomib have shown that pro-apoptotic proteins are stabilized following proteasome inhibition and contribute to the anti-cancer effect. In this report, paradoxically, we find that Bortezomib induces rapid and complete degradation of TRAF6 protein, but not mRNA, in MDS/AML cell lines and human CD34+ cells. A similar finding was observed when AML cells were treated with MG132, another proteasome inhibitor, indicating that degradation of TRAF6 is secondary to proteasomal inhibition. Interestingly, the reduction in TRAF6 protein coincides with Bortezomib-induced autophagy, as indicated by conversion of LC3B-I to LC3B-II and degradation of SQSTM1/p62, and subsequently with apoptosis in MDS/AML cells. Addition of an autophagy inhibitor (3-methyladenine [3-MA]) to Bortezomib-treated AML cells maintained TRAF6 protein expression and enhanced cell viability. Similarly, TRAF6 degradation was blocked by 3-MA when cells were treated with Rapamycin, an mTOR inhibitor and inducer of autophagy. These findings suggest that a mechanism of Bortezomib-induced cell death in myeloid malignancies involves elimination of TRAF6 protein by autophagosomes. Forced expression of TRAF6 in two AML cell lines partially blocked the cytotoxic effect of Bortezomib, suggesting that TRAF6 is an important target of Bortezomib. To determine whether loss of TRAF6 is sufficient to impede growth of MDS and AML, we used a genetic approach to inhibit TRAF6 in MDS/AML cell lines and bone marrow cells from MDS patients with deletion of chromosome 5q. RNAi-mediated depletion of TRAF6 in MDS and AML samples resulted in impaired malignant hematopoietic stem/progenitor function and rapid apoptosis. To uncover the molecular consequences following loss of TRAF6, we applied gene expression profiling and identified genes relevant to the survival of MDS and AML cells. In summary, these findings implicate TRAF6 in Bortezomib-induced cell death and in the maintenance of myeloid malignancies, and reveal a novel mechanism of TRAF6 regulation through autophagic degradation. Disclosures: Oliva: Celgene: Consultancy.

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Hh/IGF-1R/PI3K/Akt/MRP1 Pathway Induce Refractory Acute Myeloid Leukemia and Its Targeting Therpy

Blood ◽

10.1182/blood.v124.21.3612.3612 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3612-3612 ◽

Cited By ~ 4

Author(s):

Kaikai Huang ◽

Bingjie Ding ◽

Qingxiu Zhong ◽

Xuejie Jiang ◽

Xiaodong Li ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Expression ◽

Cell Lines ◽

Myeloid Leukemia ◽

Primary Cells ◽

Relapse Free Survival ◽

Free Survival ◽

Refractory Acute Myeloid Leukemia ◽

Gli 1 ◽

Acute Myeloid

Abstract Hh/IGF-1R/PI3K/Akt/MRP1 pathway induce refractory acute myeloid leukemia Background: Although a great progress has made in the treatment of acute myeloid leukemia(AML), chemoresistance and relapse due to multidrug resistance (MDR) remains a therapeutic challenge. We have proven that LBH589 combined with Bortezomib had synergistic effects on proliferation, apoptosis and sensitivity to cytotoxic drugs via AKT and NF-κB pathways in chemoresistant HL60/ADR cells and refractory AML primary cells. Using gene chip analysis, we found that among those disregulated pathways, Hedgehog(Hh) signaling pathway was probably the upstream paths which could regulate others. Aims: The purpose of the study was to investigate the expression of Hh pathway in AML including primary cells and cell lines, analyze the relationship between Hh expression and clinical prognosis. Also investigate the biological effect of Hh pathway inhibitor NVP-LDE225 in reversing drug-resistance in HL60/adriamycin-resistant(ADR) cells and refractory AML primary cells and the underlying mechanisms. Besides, animal experiments were done to verify the results in vitro. Methods: Western blot assay were used to determine the protein expression of Ptch–Smo–Gli-1–Shh in HL60 and HL60/ADR cell lines and refractory or non-refractory primary AML cells. Kaplan–Meier curves were used to estimate Relapse-free survival(RFS) and Over-all survival(OS). HL60/ADR cells and refractory primary cells were treated with adriamycin or combined with NVP-LDE225 concentration among 0-10µM). Proliferation were evaluated by 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assay and cell apoptosis were analyzed by Annexin V-FITC/PI staining through flow-cytometry(FCM). Intercellular adriamycin accumulation(MFI) were analyzed by FCM. The changes in protein levels of Gli-1–IGF-1R–p-IGF-1R–IRS-1–Akt–p-Akt–MRP-1–Bcl-2 were detected by Western blot. Besides, we used a nude mouse xenograft model to verify the anti-proliferative effects in vivo. Results: We found that the refractory primary AML cells and HL60/ADR cells correlated with higher activation of the Hh pathway, however in non-refractory primary cells or chemosensitive cell lines HL60, such activation was less pronounced. Higher protein expression of this pathway was related to higher recurrence rate and associated with poor relapse free survival (RFS) and poor overall survival (OS)(P=0.002)(a). NVP-LDE225, a potent and selective Hh inhibitor, significantly reverted resistance of adriamycin(reversal fold was 3.75), induced cell apoptosis(P=0.007), increased the intracellular adriamycin accumulation(P=0.000), decreased protein expression of p-IGF-1R, IRS-1, p-Akt, Bcl-2, MRP1 also Gli-1(b). These effects were likely to be mediated via inhibition of IGF-1R/PI3K/Akt/MRP1 pathway. Besides, in a AML xenograft mouse model, NVP-LDE225 combined with ADM resulted in significant tumor regression(P=0.002) (c). Conclusions: These findings provided evidence that targeting the Hh pathway might be a therapeutic avenue for overcoming MDR resistance in myeloid leukemia. Keywords: Hedgehog signaling, acute myeloid leukemia, IGF-1R/PI3K/Akt signaling Disclosures No relevant conflicts of interest to declare.

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Synthesis of Carboxamides Tranylcypromine Analogues as LSD1 (KDM1A) Inhibitors for AML

10.26434/chemrxiv.11860824.v1 ◽

2020 ◽

Author(s):

Teresa Borrello ◽

Chak Hin Tam ◽

Manar Shafat ◽

Stuart Rushworth ◽

Kristian Bowles ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Expression ◽

Cell Lines ◽

Myeloid Leukemia ◽

Methyl Groups ◽

Enzyme Assays ◽

Target Engagement ◽

Aryl Ring ◽

Lysine Specific Demethylase ◽

Acute Myeloid

Lysine-specific demethylase 1 (LSD1/KDM1A) oxidatively removes methyl groups from histone proteins and its aberrant activity has been correlated with cancers including acute myeloid leukemia (AML). We report a novel series of tranylcypromine analogues containing a carboxamide at the 4-position of the aryl ring and novel carbamates. These compounds were potent submicromolar LSD1 inhibitors in enzyme assays and were anti-proliferative against a panel of AML cell lines. LSD1 target engagement in cells was demonstrated through the effects on H3K4me2 protein expression, CD86, CD11b and CD14 levels

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Allosteric SHP2 Inhibitor RMC4550 Synergizes with Venetoclax in FLT3 and KIT Mutant Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-145964 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2231-2231

Author(s):

Bogdan Popescu ◽

Carlos Stahlhut ◽

Theodore C. Tarver ◽

Timothy T. Ferng ◽

Cheryl Peretz ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Tyrosine Kinase ◽

Cell Viability ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Synergistic Activity ◽

Oncogenic Signaling ◽

Subcellular Compartment ◽

Acute Myeloid

Abstract Mutations in receptor tyrosine kinases (RTK) FLT3 and KIT occur frequently in Acute Myeloid Leukemia (AML) and are associated with high risk of relapse. FLT3 tyrosine kinase inhibitors (TKI) are clinically approved in AML, but resistance is common and involves emerging clones reliant on oncogenic signaling, particularly in the RAS/MAPK pathway. Patients who relapse on FLT3 TKIs have inauspicious prognoses and no specific therapeutic options, highlighting the unmet need for effective strategies to target oncogenic signaling and improve outcomes in relapsed/refractory (R/R) AML. The protein tyrosine phosphatase SHP2 (PTPN11) is a central node in RAS/MAPK activation downstream of various RTKs, including FLT3, acting as a scaffold for adaptor proteins that promote RAS-GTP loading. Novel allosteric inhibitors are being clinically investigated in cancers with signaling activating mutations. Here, we demonstrate that the allosteric SHP2 inhibitor RMC-4550 modulates expression of pro and anti-apoptotics in FLT3 and KIT mutant AML providing rationale for combinatorial targeting of SHP2 and BCL2 as a synergistic approach. We subsequently report the preclinical efficacy of RMC-4550 and the FDA-approved, BCL2 selective inhibitor, Venetoclax combination in both in vitro and in vivo AML models. We evaluated cell viability of multiple AML cell lines treated with RMC-4550. FLT3-ITD (Molm14, MV4-11) and KIT mutant (Kasumi1, SKNO1) lines were sensitive to SHP2 inhibition. RMC-4550 maintained its efficacy in FLT3-ITD Molm14 cells with secondary mutations in FLT3 tyrosine kinase domain (TKD) and in NRAS G12C. RMC-4550 biochemically represses pERK (Figure 1A) and transcriptionally downregulates mRNA expression of DUSP6 and anti-apoptotic BCL2 and MCL1. We functionally evaluated the mitochondrial outer membrane permeabilization (MOMP) in response to SHP2 inhibition using a dynamic iBH3 profiling assay. RMC-4550 increased the overall priming and the dependency on BCL2 in both Molm14 and MV4-11 cell lines (Figure 1A). To investigate the global transcriptomic changes induced by allosteric SHP2 inhibition, we performed total mRNA sequencing on Molm14, MV4-11 and SKNO1 cell lines. GSEA analysis revealed that RMC-4550 significantly upregulated expression of genes repressed by RAS activation, downregulated MYC targets, but also dysregulated genes mediating apoptosis. The most consistently upregulated pro-apoptotic gene was BMF (fold change: 4.39, FDR<0.001). BMF is a BH3-only protein found to be sequestered to motor filaments that, in response to cellular damage signals, is translocated in the cytoplasm and binds pro-survival Bcl2 proteins. The BMF transcript upregulation was confirmed by qPCR and western blot analysis showed a marked overexpression of the BMF protein level upon SHP2 inhibition, particularly in the cytoplasmic subcellular compartment (Figure 1B). We next treated Molm14, MV4-11, Kasumi and SKNO1 lines with incremental doses of RMC-4550 and Venetoclax in an 8x8 combination matrix to assess the synergy of the two compounds using cell viability and apoptosis readouts. The assay showed highly synergistic activity in both FLT3-ITD and KIT lines. Remarkably, we noted a potent synergy in Molm14 cells with concurrent mutation in NRAS G12C (Figure 1C). In a Molm14 cell line xenograft model, we demonstrated that the combination of RMC-4550 (30 mg/kg) and Venetoclax (100 mg/kg) administered orally 5 times a week for 28 days significantly decreased leukemia burden and improved survival (p<0.001) compared to control and single agents (Figure 1D). In a FLT3-ITD AML patient-derived xenograft (PDX) model, the combination of RMC-4550 and Venetoclax markedly decreased %hCD45 in both cardiac blood and spleen of NSGS mice compared to vehicle-treated control (Figure 1E). Supporting a potential therapeutic index for the combination, RMC-4550 and Venetoclax strongly inhibited colony formation in FLT3 AML primary samples compared to samples from healthy volunteers. Collectively, our data suggest that SHP2 inhibition increases the apoptotic dependency on BCL2 through up-regulation of the pro-apoptotic BMF, a mechanistic rationale to synergistically inhibit both targets. We provide preclinical evidence that co-targeting SHP2 and BCL2 is a potential effective therapeutic strategy in RTK-driven AML. Figure 1 Figure 1. Disclosures Stahlhut: Revolutions Medicine: Current Employment, Current equity holder in publicly-traded company. Smith: Daiichi Sankyo: Consultancy; Amgen: Honoraria; AbbVie: Research Funding; Revolutions Medicine: Research Funding; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

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Growth Inhibitory Function Of a Novel T-LAK Cell-Originated Protein Kinase Inhibitor On FLT3-ITD Positive Acute Myeloid Leukemia (AML) Cells

Blood ◽

10.1182/blood.v122.21.1459.1459 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1459-1459

Author(s):

Houda Alachkar ◽

Jae-Hyun Park ◽

Makoto Nakakido ◽

Alex Wood ◽

Gordana Raca ◽

...

Keyword(s):

Protein Kinase ◽

Cell Viability ◽

Cell Lines ◽

Myeloid Leukemia ◽

Kinase Inhibitor ◽

U937 Cells ◽

Transfected Cells ◽

Lak Cell ◽

Acute Myeloid

Abstract T-LAK cell-originated protein kinase (TOPK/PBK), a serine-threonine mitogen-activated protein kinase kinase is highly expressed and correlated with more aggressive disease in several types of cancer, but is undetectable in normal tissues except the testis and fetal tissues. TOPK is up-regulated in a variety of hematologic malignancies including acute myeloid leukemia (AML) and may be involved in disease pathogenesis. To investigate the role of TOPK in AML and to develop the rationale for therapeutic targeting, we initially examined the expression level of TOPK protein in AML cell lines by western blot analysis. TOPK expression was detected at high levels in 8 out of 11 AML cell lines. Importantly, TOPK was not detected in mobilized, peripheral blood-derived stem cells from healthy donors. To determine the role of TOPK in AML, we utilized a loss of function approach in two AML cell lines (MV4-11 and U937 cells) and assessed cell viability and apoptosis by MTS and Annexin/PI staining assays, respectively. Cells transfected with TOPK-siRNA showed significant decrease in cell viability (∼70%, P<0.001, both cell lines) and significant increase in apoptosis 48 hours following transfection compared to cells transfected with control-siRNA. A small molecule compound that inhibits TOPK kinase is currently undergoing pharmaceutical development for cancer treatment. We treated nine AML cell lines with the compound for 48 hours and assessed cells viability by MTS assay. Decrease in cell viability following treatment with increasing doses of the compound was noted with variable sensitivities among AML cell lines. In addition, the compound showed enhancement of cell differentiation assessed by CD11b staining in U937 cells. Cell lines with FLT3 mutations (MV4-11, MOLM13 and KOCL-48) were significantly more sensitive to the treatment compared to cell lines with unmutated FLT3. We next treated cells with increasing doses of the compound and assessed cells viability and apoptosis by MTS and flow cytometry assays. Following the treatment with the compound, IC50 was <10nM for MV4-11 and MOLM13 cells (FLT3-ITD positive cell lines), and >20nM for U937 and KG1 (FLT3-ITD negative cell lines). 40 nM of the compound induced apoptosis by ∼80% in MV4-11 and MOLM13 cells compared to 49% in U937 and 9% in KG1 cells, at 48 hours following treatment. We also validated the anti-leukemia activity in primary blasts from patients with AML (n=2). FLT3-ITD positive blasts had an IC50 of ∼15nM and showed 40% increase in apoptosis following treatment with 20nM of the compound. To gain a mechanistic insight into why FLT3-ITD mutant cells are preferentially sensitive to this TOPK inhibitor, we performed a gene expression profile microarray analysis on MV4-11 cells treated with 20nM of the compound or transfected with TOPK siRNA in comparison with untreated cells and cells transfected with control-siRNA. We observed significant downregulation in genes involved in cell cycle control pathways in the signatures associated with the compound-treated and TOPK siRNA-transfected cells. Interestingly, FLT3 was among the significantly downregulated genes in the compound treated cells (∼80%) and in TOPK-siRNA transfected cells (∼30%). We then examined the activity and the expression levels of FLT3 protein following the treatment. Consistently, we found that both the phospho-FLT3 and the total FLT3 protein levels were completely depleted in cells treated with 10, 20 and 40 nM of the compound as early as 16 hours following treatment. In conclusion, TOPK is highly expressed in AML and may act as novel therapeutic target. A novel TOPK kinase inhibitor exhibits preferential cytotoxicity to FLT3-ITD mutated AML cells, possibly through inhibition of FLT3 protein expression. Although further research is needed to determine the mechanism by which the compound inhibits FLT3 protein expression, this novel compound may represent a new targeted therapy for this adverse risk subset of patients with AML. Disclosures: Matsuo: OncoTherapy Science, Inc.: Employment. Nakamura:Oncotherapy Science. Ltd.: share holder Other.

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Obatoclax reduces cell viability of acute myeloid leukemia cell lines independently of their sensitivity to venetoclax

Hematology Transfusion and Cell Therapy ◽

10.1016/j.htct.2021.01.004 ◽

2021 ◽

Author(s):

Keli Lima ◽

Hugo Passos Vicari ◽

Jorge Antonio Elias Godoy Carlos ◽

Jean Carlos Lipreri da Silva ◽

Lorena Lobo de Figueiredo-Pontes ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Viability ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Acute Myeloid Leukemia Cell ◽

Leukemia Cell Lines ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

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Clinical Impact and Therapeutic Opportunity of Insulin Receptor Substrates 1/2 in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-119156 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1512-1512

Author(s):

Juan Luiz Coelho-Silva ◽

Diego Antonio Pereira-Martins ◽

Josiane Lilian Schiavinato ◽

Eduardo Magalhães Rego ◽

João Agostinho Machado-Neto ◽

...

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukemia ◽

Cell Viability ◽

Cell Lines ◽

Myeloid Leukemia ◽

Annexin V ◽

Ros Production ◽

Insulin Receptor Substrates ◽

Induced Apoptosis ◽

Acute Myeloid

Abstract Background: The identification of biological and clinical prognostic factors in acute myeloid leukemia (AML) allowed the definition of patient subgroups and the realization of risk-adapted and targeted treatment strategies. Insulin-like growth factor 1 receptor/Insulin Receptor Substrates (IGF1R/IRS) pathway plays an important role in the development of neoplasia. IRS1/2 activates AKT/mTOR and MAPK pathways, through their interaction with PI3K and GRB2, culminating in increasing cell proliferation. NT157 is an allosteric inhibitor of IGF1R-IRS1/2 signaling that showed antineoplastic effects in preclinical studies of solid tumors. However, IRS1/2 clinical function and NT157 effects were not assessed in AML. Aims: To investigate IRS1 and IRS2 mRNA expression in AML patients and their impact in clinical outcomes, and to analyze the effects of the NT157 in AML cell lines. Material and methods: Comparison of IRS1 (probe nº 204686) and IRS2 (probe nº209184_s) expression from 581 AML patients and 8 CD34+ cells from healthy subjects were analyzed using data from Amazonia! Platform. For survival analysis, IRS1 and IRS2 mRNA expression levels from 173 AML patients (92 male - median age 58 years [range: 18-65]) were obtained from TCGA AML study available online on CBioPortal for Cancer Genomics. NB4, NB4-R2, Kasumi-1 and THP1 cell lines were submitted to NT157 (0.5, 1.0, 2.0, 4.0, 8.0 or 16 µM) 72 hours and evaluated for cell viability (MTT assay), apoptosis (Annexin V/PI), cell cycle (PI), ROS production (DCFDA), mitochondria staining (MitoTracker), and protein expression/activation (western blot). Bone marrow mononuclear cells (BMMC) were obtained from 4 AML patients at diagnosis and submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA, Mann-Whitney or Kruskal-Wallis and Spearman correlation tests, as appropriate. For survival analysis, Kaplan-Meyer curves were compared with the log-rank test. Cox regression analysis was also applied. Results: IRS1 expression, but not IRS2, predicted outcomes. Reduced IRS1 expression showed poorer disease-free survival (DFS) (survival median time [MT]: 10.1 months [mos] vs. 28.4 mos, P<0.001; Hazard ratio [HR]: 0.51 [CI95:0.32 - 0.79]) and overall survival (OS) (MT: 14.5 mos vs. 27.4 mos, P=0.009; HR: 0.61 [CI95:0.42 - 0.88]). IRS1 expression independently predicted poorer DFS (HR: 0.59 [CI95: 0.36 - 0.79]; P= 0.03) using cytogenetic risk stratification, age and leukocytes as confounders. Of note, IRS1 level was positively correlated with proapoptotic CD27 (r=0.51; P<0.001) and with IL17RA (r=0.62; P<0.001) related to CD34 cell differentiation. IRS2 expression was upregulated in AML harboring t(15;17) (n=36; P<0.01) and inv(16) (n=37; P=0.01) in comparison to CD34+. In NB4, NB4-R2 and Kasumi-1 cells, NT157≥0.5µM reduced cell viability (P<0.05) and increased apoptosis (P<0.05). The mean percentage of annexin V+ cells for control, NT157 2.5, 5 and 10µM were 11, 47, 73 and 75% for NB4, 11, 41, 69 and 75% for NB4-R2 and 17, 45, 61 and 64% for Kasumi-1, respectively. In TP53-null cell line THP1, NT157 reduced cell viability at doses higher than 2µM (P<0.05) and induced apoptosis at 10µM (9.1 vs. 25%; P<0.05). NT157 induced ROS production in NB4 (fold-increase of mean fluorescence intensity [MFI]: 25.8 and 24.8), NB4-R2 (MFI: 26.7 and 31.4), Kasumi-1 (MFI: 5.8 and 6.6) and THP1 (MFI: 1.8 and 4.1) at 5 and 10µM (all P<0.05) and increased mitochondrial mass in NB4 (MFI: 3.9 and 3.7), NB4-R2 (MFI: 2.6 and 2.9), Kasumi-1 (MFI: 3.2 and 4.7) and THP-1 (MFI: 2.6 and 2.2) (all P<0.05). NT157 also modulated cell cycle progression, as evidenced by G2/M arrest in THP-1 and sub-G0/G1 in other cell lines (P<0.05). The IGF1R-IRS1/2 inhibitor NT157 reduced activation/expression of IGF1R (Tyr1135), IRS1/2 (Tyr612), AKT1/2/3 (Ser473), P70S6K (Thr421/Ser424), 4EBP1 (Thr70), ERK1/2 (Thr185/Tyr187) and induced DNA damage (increased γH2AX). NT157 did not induce autophagy, as demonstrated by non-degradation of p62 and lack of conversion of LC3BI into LC3BII in cell lines tested. NT157≥0.5 µM reduced cell viability and induced apoptosis in BMMC from 4 AML patients in a dose dependent manner (P<0.05). Conclusions: In AML, downregulation of IRS1 predicted dismal prognosis and the IGF1R-IRS1/2 inhibitor NT157 exerted an antineoplastic activity, downregulated PI3K/AKT and MAPK signaling. IRS1/2 arises as a promising therapeutic target for AML patients. Disclosures No relevant conflicts of interest to declare.

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The Survivin Suppressant YM155 (Sepantronium Bromide) Has Potent Pre-Clinical Activity Against Pediatric Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.3957.3957 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3957-3957

Author(s):

Erica B. Little ◽

Amanda M. Smith ◽

Andjelija Zivanovic ◽

Alfred K.S. Liu ◽

Shannon Waldron ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Viability ◽

Cell Lines ◽

Myeloid Leukemia ◽

Annexin V ◽

Clinical Activity ◽

Pediatric Aml ◽

Acute Myeloid ◽

Sepantronium Bromide

Abstract Despite aggressive chemotherapy and a high remission induction rate, approximately one third of children with acute myeloid leukemia (AML) relapse. Therefore, novel and more effective treatments are urgently needed. Survivin is an inhibitor-of-apoptosis protein that plays a key role in regulating cell division, proliferation and apoptosis (Altieri et al, Nat Rev Cancer, 2008). Furthermore, high expression of survivin and its splice variants have been shown to be associated with poor clinical outcome in AML (Moore et al, ASH Abstract 3555, 2011; Carter et al, Blood, 2012). The small-molecule survivin suppressant YM155 (sepantronium bromide) has been demonstrated to have pre-clinical activity against a range of solid cancers and leukemias, although data in AML is limited, particularly in pediatric models. Therefore, we undertook a comprehensive pre-clinical evaluation of YM155 in AML, with a focus on pediatric disease. When tested in vitro against a diverse panel of 9 AML cell lines, YM155 potently inhibited cell viability with a median IC50 of 0.03 µM (range 0.001 - 0.680 µM; Table 1). All 4 pediatric cell lines tested (Kasumi-1, MV4-11, THP-1 and CMK) were particularly sensitive to YM155, with IC50 values in the range of 0.010 - 0.053 µM. Of note, YM155 had generally similar in vitro efficacy to daunorubicin, but was more potent than cytarabine in 7 of the 9 cell lines.Table 1In vitro sensitivity of AML cell lines to YM155, cytarabine and daunorubicin, as measured by 72 hour cell viability (resazurin reduction) assays. Values indicate median IC50(µM) from at least 3 independent experiments. *Pediatric AML cell lines. NS, not significant.AML Cell LineCharacteristic FeatureYM155CytarabineDaunorubicinP value (YM155 vs. Cytarabine)HL-60Complex karyotype0.0010.4810.019NSKasumi-1*t(8;21)(q22;q22)0.0100.1120.0220.011RUNX1-CBFA2T1c-KITmut(N822K)ML-2t(6;11)(q27;q23)0.0100.0620.0050.027OCI/AML3NPM1mut0.011>100.020<0.001MV4-11*t(4;11)(q21;q23)0.0301.2630.004<0.001MLL-AF4FLT3-ITDTHP-1*t(9;11)(p21;q23)0.0367.0440.027<0.001CMK*AML assoc. with Down Syndrome0.0530.4280.078<0.001HELAML M60.5310.0780.1310.011ME-1inv(16)(p13q22)0.6800.3900.298NSCBFB-MYH11 Cell cycle analyses demonstrated concentration-dependent increases in the sub-G1 fraction of cell lines treated with YM155, suggestive of apoptosis. An apoptotic mechanism of cell death was confirmed by an increase in annexin V positivity, measured by flow cytometry. Consistent with the proposed mechanism of action, YM155 treatment also caused down-regulation of total survivin protein expression in AML cell lines. Furthermore, in vitro assays demonstrated that YM155 was cytotoxic against a panel of 7 primary, diagnostic bone marrow samples from children with AML (median age 4.8 years, range 1.3-15.7). Patient samples were obtained from the Queensland Children's Tumour Bank and had diverse cytogenetic and molecular characteristics, including CBF-AML, monosomy 7, MLL gene rearrangements and FLT3-ITD. Similar to experiments in established cell lines, YM155 treatment caused an apoptotic response, as evidenced by induction of annexin V positivity. In conclusion, YM155 has potent pre-clinical activity against a broad panel of AML cell lines and patient samples. Pediatric AML appears to be particularly sensitive to YM155. These data suggest that YM155-mediated inhibition of survivin is a potentially beneficial therapeutic strategy for AML, particularly childhood disease, and warrants further pre-clinical and clinical evaluation. Disclosures: No relevant conflicts of interest to declare.

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