Resistant Acute Myeloid Leukemia Cell Lines Research Articles

Galectin-9 has non-apoptotic cytotoxic activity toward acute myeloid leukemia independent of cytarabine resistance

Acute myeloid leukemia (AML) is a malignancy still associated with poor survival rates, among others, due to frequent occurrence of therapy-resistant relapse after standard-of-care treatment with cytarabine (AraC). AraC triggers apoptotic cell death, a type of cell death to which AML cells often become resistant. Therefore, therapeutic options that trigger an alternate type of cell death are of particular interest. We previously identified that the glycan-binding protein Galectin-9 (Gal-9) has tumor-selective and non-apoptotic cytotoxicity towards various types of cancer, which depended on autophagy inhibition. Thus, Gal-9 could be of therapeutic interest for (AraC-resistant) AML. In the current study, treatment with Gal-9 was cytotoxic for AML cells, including for CD34+ patient-derived AML stem cells, but not for healthy cord blood-derived CD34+ stem cells. This Gal-9-mediated cytotoxicity did not rely on apoptosis but was negatively associated with autophagic flux. Importantly, both AraC-sensitive and -resistant AML cell lines, as well as AML patient samples, were sensitive to single-agent treatment with Gal-9. Additionally, Gal-9 potentiated the cytotoxic effect of DNA demethylase inhibitor Azacytidine (Aza), a drug that is clinically used for patients that are not eligible for intensive AraC treatment. Thus, Gal-9 is a potential therapeutic agent for the treatment of AML, including AraC-resistant AML, by inducing caspase-independent cell death.

Abstract B019: ASO-mediated NRAS knockdown overcomes gilteritinib late resistance in FLT3-AML

Abstract The internal tandem duplication of the FLT3 receptor (FLT3-ITD) leads to constitutive receptor activation and is one of the most common mutations (~30%) in acute myeloid leukemia (AML), resulting in poor prognosis. Multiple FLT3 inhibitors (FLT3i) have been developed, including the potent FDA-approved FLT3i, gilteritinib. However, AML patients only respond to gilteritinib for approximately 6 months due to the emergence of drug resistance. Through an approach that integrated genomic, metabolomic, proteomic, and phosphoproteomic analyses with complementary genetic and pharmacologic screens, we demonstrated that gilteritinib resistance occurs in a stepwise manner with two distinct phases of early and late resistance (Joshi et al., Cancer Cell, 2021). While gilteritinib efficiently eliminates leukemia cells in the blood, residual cells survive within the bone marrow, where they are protected by extrinsic microenvironmental factors fostering early resistance. These lingering cells eventually develop intrinsic mutations in NRAS that allow them to resist gilteritinib treatment, leading to late resistance. Acquisition of mutations in the RAS pathway, particularly NRAS, are a common mode of late resistance (~11%) for many patients that relapse on FLT3i. Developing therapeutics to block NRAS activity remains a challenge with the lack of direct small-molecule inhibitors. Indirect approaches to inhibit mutant NRAS/MAPK pathway signaling have also not met much clinical success. Hence, therapeutic nucleic acid-based approaches, including antisense oligonucleotides (ASOs), may offer an alternative approach to target NRAS-driven cancers. We evaluated the effectiveness of a novel ASO targeting NRAS mRNA to restore gilteritinib sensitivity in resistant cultures in vitro and in primary AML patient samples ex vivo. We first assessed the potency of the NRAS ASO in both gilteritinib-sensitive and late resistant MOLM-14 AML cell lines (N=9) that expressed wild-type or mutant NRAS (G12S or G12D) via free uptake of ASO. NRAS expression was evaluated by qPCR after 48 hours. ASO treatment down-regulated mutant NRAS mRNA expression in a dose-dependent manner with an IC50 of < 1 µM and decreased NRAS protein levels. No changes in NRAS mRNA or protein levels were observed with non-targeting ASO control. Decreased NRAS mRNA expression restored sensitivity to gilteritinib in late-resistant cultures, leading to decreased cell viability and increased apoptosis. A similar trend was observed in primary AML cells from patients (N=2) that relapsed on gilteritinib, where reduction of approximately 50% of mutant NRAS mRNA robustly sensitized patient cells to gilteritinib. In contrast, the combination of gilteritinib and the MEK inhibitor trametinib was not as effective in either cell cultures or patient samples. Our results suggest that ASO-mediated knockdown of NRAS may circumvent the development of resistance and improve the durability of FLT3i. Citation Format: Sunil K. Joshi, Janét Pittsenbarger, Xiaolin Luo, Hadi Maazi, Alexey Revenko, Brian J. Druker, Elie Traer. ASO-mediated NRAS knockdown overcomes gilteritinib late resistance in FLT3-AML [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B019.

The Multi-CDK Inhibitor Dinaciclib Shows Synergistic Activity with the BET Inhibitor PLX51107 and Reverses BET Resistance through Inhibition of Canonical Wnt Signaling in Acute Myeloid Leukemia (AML)

PLX51107 is a novel BET inhibitor (BETi) with antineoplastic activity in pre-clinical models of both aggressive B-cell malignancies and AML. Differentiating it from other BET inhibitors currently under investigation, PLX51107 has a unique binding site in the acetylated lysine binding pocket of BRD4. Through interaction with pTEFb, which is comprised of CDK9 and several cyclin T1 heterodimers, BRD4 promotes phosphorylation of RNA polymerase II, ultimately influencing gene transcription. Dinaciclib is a potent inhibitor of multiple cyclin dependent kinases including CDK9 and has been shown to have pre-clinical activity in AML. Therefore, we hypothesized that PLX51107 would have synergistic activity when combined with dinaciclib. We found synergistic effects on proliferation in primary AML cells when PLX51107 was combined with dinaciclib in stromal co-cultures across a range of dose combinations below the IC50 of either compound alone. Synergistic activity was also present in cytokine support culture conditions, colony forming assays, and synergy assays utilizing COMbenefit software. Interestingly, the highly conserved canonical β-catenin dependent Wnt signaling pathway is a known BETi resistance mechanism in AML. Therefore, destruction of β-catenin and inhibition of Wnt signaling could represent a novel therapeutic target to circumvent development of BET-mediated resistance. We found dinaciclib treatment reduced protein levels of total β-catenin in primary AML cells and AML cell lines (Figure 1A). Phosphorylated β-catenin and non-phosphorylated "active" β-catenin protein levels were also reduced following dinaciclib treatment (Figure 1A). In addition, mRNA expression of multiple components of the Wnt pathway, including the Wnt co-receptor LRP6, AXIN2, FZD1, and many Wnt target genes were significantly decreased following dinaciclib and combination treatment. Moreover, in a luciferase based Wnt reporter cell line, we found dinaciclib significantly reduced Wnt3a induced luciferase activity, suggesting a direct effect on β-catenin mediated transcription of Wnt target genes. Collectively, this data suggests dinaciclib has multiple inhibitory effects on canonical β-catenin dependent Wnt signaling. We found combination treatment of PLX51107 and dinaciclib produced synergistic reduction in expression of both Wnt signaling components and Wnt target genes. Conversely, β-catenin and Wnt target gene expression levels were either not significantly changed or even increased when treated with PLX51107 alone. In order to examine the effects of dinaciclib in the setting of BET resistance, we developed PLX51107 resistant MOLM-13 and MV4-11 AML cell lines via serial drugging. These resistant cell lines remained sensitive to dinaciclib treatment and to cytotoxic chemotherapy drugs including cytarabine and etoposide while demonstrating resistance to PLX51107 via Annexin PI and MTS assays. We found sensitivity to dinaciclib treatment in these resistant cell lines remained unaffected by BETi resistance and continued to demonstrate inhibition of Wnt signaling, similar to the effects seen in parental cells. Therefore, dinaciclib represents a novel strategy to overcome potential therapy emergent BETi resistance. We next examined the efficacy of PLX51107 in combination with dinaciclib in vivo, utilizing luciferase labeled MOLM-13 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 42 days) compared to vehicle treated control animals (median 28 days, p< 0.001). Weekly intraperitoneal 10 mg/kg dinaciclib did not significantly improve survival (median 30 days, p=0.09) in MOLM-13 xenograft mice. However, mice receiving combination therapy survived a median of 56.5 days, significantly longer than with either drug alone (p<0.05) (Figure 1B). We also found decreased disease burden by bioluminescence and by histopathology in combination treated animals, compared to both vehicle and single agent controls. In conclusion, we have demonstrated in vitro and in vivo synergistic activity of the BET inhibitor PLX51107 in combination with dinaciclib in pre-clinical models of AML and have identified novel inhibition of Wnt / β-catenin signaling by dinaciclib, thereby providing a strategy to circumvent resistance to BET inhibitors in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Cholesterol Homeostasis Pathway to Overcome Acquired Cytarabine Resistance in Acute Myeloid Leukemia

The current backbone of Acute Myeloid Leukemia (AML)therapy is treatment with the cytidine analogue Ara-C and the anthracycline daunorubicin. Despite a high rate of initial remissions, a substantial fraction of patients with AML relapse and acquire resistance to Ara-C. In acquired resistance, there is an initial therapy response, but resistance develops over time. To uncover the acquired drug resistant mechanisms, we utilized two cytarabine (Ara-C) resistant cell lines - THP1 and U937 developed from the parental respective cell lines (kind gift from Dr. Martin Michaelis, University of Kent). In-vitro cytotoxicity assay confirmed the cytarabine resistance in THP1 cell line (THP1 Ara-C R IC50: 1457µM; THP1 parental IC50: 56 µM) and U937 cell line (U937 Ara-C R IC50: 1578.3 µM and U937 parental IC50: 0.14 µM). THP1-AraC-R was marginally cross resistant to both DNR (IC50 Parent -0.19µM, AraC 0.26µM) and ATO (IC50 Parent -2.1µM, AraC >4µM). To identify the factors associated with Ara-C drug resistance, we subjected both the cell lines along with their parental counterparts for transcriptomic profiling. Several protein coding genes were differentially expressed in both the cell lines. A total of 845 genes were upregulated and 268 genes were downregulated in THP1 Ara-C R cell line while 344 genes were upregulated, and 647 genes were downregulated in U937 Ara-C R cell line. To unravel the pathways associated with Ara-C resistance, the differentially expressed genes were subjected to Gene Set Enrichment Analysis (GSEA). The pathway enrichment showed Cholesterol Homeostasis pathway to be highly enriched in THP1 (NES: 2.17 & FDR q-value: 0) and U937 (NES:1.25 & FDR q-value:0.13) Ara-C resistant cell lines in comparison to the respective parental cell lines. To address the role of Cholesterol Homeostasis pathway in mediating chemoresistance in these two Ara-C resistant AML cell lines (THP1, U937) we screened few selected pharmacological inhibitors targeting different genes in the Cholesterol Homeostasis pathway [HMG Co-A reductase (HMGCR) the rate limiting step in Cholesterol metabolism, Squalene synthase (SQS), an intermediate enzyme in the pathway of Cholesterol synthesis and HMG Co-A synthase (HMGCS1) catalyzes the condensation of acetyl Co-A with aceto-acetyl Co-A to form HMGCo-A ]. Among all the inhibitors screened, Hymeglusin, an inhibitor of HMGCS1, re-sensitized only the THP1 Ara-C resistant cell with good synergy (combinatorial Index for THP1 Ara-C cell line 0.68 and 0.2 with Ara-C). These results demonstrate the dependence of leukemic cells on the Cholesterol Homeostasis pathway to mediate cytarabine resistance and pharmacological inhibition of this pathway can result in the reversal of cytarabine resistance.

Abstract 3973: Transcriptomic profiling of in-vitro modelled drug resistant AML cell lines unravel metabolic and stemness gene signatures

Abstract Disease recurrence is the major cause of treatment failure and mortality in acute myeloid leukemia (AML). A small fraction of leukemic cells that withstand chemotherapeutic insult can rewire cellular processes to re-emerge with lethal disease. Approaches to decipher drug resistance mechanisms in these drug persister cells can pave the way for newer therapies and improve survival in patients. To uncover drug resistant mechanisms that are clinically relevant, we generated in-house Cytarabine (AraC) and Arsenic trioxide (ATO) resistant cells from naïve MV4-11 AML cell line by incremental dose exposure method. Single cell clones were generated from AraC (MV411-AraC-R)/ATO (MV411-ATO-R) drug tolerant cells. Both the AraC-R and ATO-R clones were resistant to AraC (600-fold) and ATO (2-fold) respectively compared to the naïve cells. AraC-R cells were marginally cross resistant to daunorubicin (DNR) and ATO. Interestingly, the ATO-R cells were highly cross resistant to both AraC (600-fold) and DNR (4-fold) demonstrating altered drug resistance mechanism. We next subjected the AraC-R, ATO-R and the naïve cells to RNAseq analysis to identify transcriptomic changes post resistance development. 1218 transcripts in AraC-R cells and 7654 transcripts in ATO-R cells were differentially expressed compared to the naïve cells, suggesting ATO exposure alters the transcriptome drastically compared to AraC. We performed gene set enrichment analysis (GSEA) with these differentially expressed gene signatures. AraC-R gene signatures were highly enriched (NES-2.8 &amp; 2.0 FDR-0.00) for oxidative phosphorylation and mitochondrial related signatures, consistent with previous report suggesting AraC exposed residual cells are highly enriched for oxidative phosphorylation and are not enriched for stemness gene signatures (Farge et al). ATO-R gene signatures were positively enriched (NES- 2.0 &amp; 1.4 FDR 0.005 &amp; 0.03) for Leukemic stem cell gene signatures (Eppert_LSC and Gentles_LSC) and relapse (NES- 2.0 &amp; 1.5 FDR 0.0 &amp; 0.01) gene signatures (CALCRL_RIC and Hackles_relapse) and negatively enriched for mitochondrial gene signatures showing reduced reliance on mitochondrial metabolism. Since AraC-R cells showed high dependency on mitochondrial metabolism, we treated these cells with venetoclax (VEN) a known oxphos inhibitor for 48 hours and assessed the viability. Ven markedly sensitized the AraC-R cells compared to naïve cells (IC50 13nM and 323nM), while ATO-R cells were markedly resistant to Ven (IC50 8049nM) corroborating our transcriptomic findings. These finding suggest transcriptome profiling of in-vitro modelled drug resistant cell lines can mimic clinically relevant drug resistant mechanism and could aide in identifying alternate therapies to circumvent disease relapse. Further metabolomic characterization and mechanistic studies are ongoing to substantiate these findings. Citation Format: Raveen Stephen Illangeswaran, Daniel Zechariah Jebanesan, Kezia Kanimozhi Sivakumar, Rakhi Thalayattu Vidhyadharan, Vikram Mathews, Shaji Ramachandran Velayudhan, Poonkuzhali Balasubramanian. Transcriptomic profiling of in-vitro modelled drug resistant AML cell lines unravel metabolic and stemness gene signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3973.

Transcriptomic Signitures of Azacitidine (AZA) and Decitabine (DAC) Resistance in MDS and CMML

INTRODUCTION: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are heterogeneous myeloid neoplastic disorders characterized by ineffective hematopoiesis leading to cytopenias and increased risk of transformation to acute myeloid leukemia (AML) . The hypomethylating agents (HMA) azacitidine (AZA) and decitabine (DAC) improve the natural history of MDS and CMML patients (Fenaux et al 2009). However, over half of the cases experience primary failure defined by a lack of response to HMA treatment which is associated with poor prognosis and a median survival of 4-6 months (Garcia-Manero et al. 2016, Jabbour et al. 2010). The highly heterogeneous pathophysiology of myeloid neoplasms and poorly understood mechanisms underlying therapeutic action of HMAs pose substantial challenges in understanding the biology of HMA failure in MDS and CMML.METHODS: We established a cohort of baseline bone marrow (BM) cells that were collected from 17 CMML and 34 MDS patients with excessive blasts (MDS-EB) prior to their HMA based therapies (Figure 1). RNA-Seq based transcriptomic analysis was performed in CD34+ BM hematopoietic stem and progenitor cells (HSPCs) of patients, known to be the cellular origin of these diseases, to identify biological signatures of primary HMA resistance.RESULTS: Similar to the recent reports regarding the lack of common gene expression signatures in HMA resistant AML cell lines (Leung et al. 2019) , RNA-Seq in patient cohort detected fewer than 0.5% of the total number of differentially expressed genes in non-responders in common for both AZA and DAC. Of note, all the AZA and a large portion of DAC associated genes with down-regulations in non-responders encode immunoglobulins, which is consistent with several recent findings indicating impaired differentiation B cells in association with unfavorable outcomes in MDS and CMML (Ribeiro et al. 2006 and Kahn et al. 2015). We performed flow cytometry analysis in BM cells available for 13 patients prior to start of HMA treatments (5 non-responders and 8 responders), and detected that HMA non-responders had a strong tendency (P=0.07) of decreased baseline frequencies of B cells in their BM than HMA responders.GSEA analysis based on leading edge genes identified over 200 and 300 biological signaling pathways to be associated with AZA and DAC failure respectively, with 78 pathways commonly correlated with treatment failure of both drugs (28 up-regulated and 60 down-regulated, Table 1). Clustering of these commonly altered pathways based on biological functions revealed that most of them are known to have a role in MDS and CMML pathogenesis and/ or drug resistance. For instance, neurotransmitter, olfactory pathways, and associated G-protein coupled receptor signals was recently reported to play a role in regulating the maintenance and differentiation of BM HSPCs (Shao et al. 2021 and Shim et al. 2013), whereas cell junction signaling that involves integrins and increased MAP2K-MAPK signaling were also reported to be associated with AZA resistance in MDS and CMML (Unnikrishnan et al. 2017).Among commonly down-regulated biological pathways in non-responders of AZA and DAC, there were clusters of immunoglobulin-associated signals, protein translation regulatory pathways that involve ribosomal proteins, cell cycle signaling, respiratory chain, and Golgi transport signals. Decreased expression of ribosomal proteins and related impairment of ribosomal functions were known mechanism in MDS and CMML development (Ebert et al. 2008 and Schneider et al. 2016). Furthermore, decreased respiratory chain and Golgi transport signals were also identified by transcriptomic investigation in the DAC resistant TF1-RES cell lines.In addition to the commonly altered signals for both AZA and DAC resistance, innate immune signaling pathways including interferon and toll-like receptor signals were significantly up-regulated in DAC non-responders but down-regulated in AZA non-responders.CONCLUSIONS: In this study, the transcriptomic data between responders and non-responders to AZA and DAC were investigated separately, thereby drug-specific as well as the common biomarkers associated with treatment failures of both drugs could be identified. The relatively low proportion of genes and pathways shared by AZA and DAC non-responders suggest the difference underlying biological mechanisms of AZA and DAC failure. [Display omitted] DisclosuresSasaki: Novartis: Consultancy, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees. Wei: Daiichi Sanko: Research Funding.

Novel Oxidative Phosphorylation Inhibitor IACS-010759 Induces AMPK-Dependent Apoptosis of AML Cells

Acute myeloid leukemia (AML) cells frequently adjust to increased energy/substrate demand under stress conditions in the bone marrow microenvironment. Recent studies indicate that AML cells including leukemia-initiating cells are highly dependent on oxidative phosphorylation (OxPhos) for survival, while normal hematopoietic stem cells predominantly utilize glycolysis for energy homeostasis.The aim of this study is to assess the biomarkers of anti-leukemia efficacy of the first-in-class oxidative phosphorylation inhibitor (OxPhosi) IACS-010759. IACS-010759 at nanomolar concentrations blocks cellular respiration through inhibition of complex I of the electron transport chain (Molina et al., AACR 2016), is orally bioavailable and is currently in Phase 1 clinical trial in AML (NCT #02882321).We utilized primary AML samples from patients with newly diagnosed or relapsed/refractory AML (11 sensitive and 3 resistant to IACS-010759) as well as OxPhosi-sensitive OCI-AML3 and -resistant MOLM13 AML cell lines . To identify transcript abundances for the transcriptome profiling, Cap Analysis of Gene Expression (CAGE)-Seq analysis has been conducted. CAGE-Seq identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression. CAGE-Seq analysis identified recurrent baseline differences of TSS gene expression between IACS-010759-sensitive and -resistant primary AML samples; 4 genes were higher and 124 genes were lower in sensitive samples compared to resistant samples (false positive rate; FDR<0.05). In IACS-010759 sensitive OCI-AML3 cells the expression levels of 1,623 genes were higher and 1,821 were lower than in resistant MOLM13 cells (triplicate, FDR<0.05). Based on these transcriptome differences, the Ingenuity Pathway Analysis (IPA) bioinformatics platform highlighted consistent overexpression of 4 factors in IACS-010759-resistant AML primary samples and cell line compared to the sensitive AML. These include mammalian target of rapamycin (mTOR) kinase, a positive regulator of cell metabolism; Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2) that enhances kinase-mediated activation; erythropoietin (EPO), a positive regulator of Jak-Stat pathway; and IL3, a potent growth-promoting cytokine. These findings indicate that the basal metabolic energetic capacity might affect the sensitivity of AML cells to IACS-010759. AMPK is a crucial cellular energy sensor and regulates energy metabolism through mTOR inhibition. We therefore studied the role of energy metabolism regulator AMP-activated protein kinase (AMPK) and of mTOR in anti-leukemia efficacy. To validate the role of AMPK, we utilized AMPK knockdown (sh AMPK) OCI-AML3 cells, and examined the sensitivity against IACS-010759, comparing to parental shC OCI-AML3 cells. By immunoblotting, IACS-010759 induced significant increase of p-AMPK in parental OCI-AML3 cells along with decrease of mTOR downstream target phospho- (p-) 4EBP1. OCI-AML3 stably infected with sh AMPK predictably overexpressed p-4EBP1 which was not modulated by IACS-010759. sh AMPK OCI-AML3 cells were more than 10-fold less sensitive against IACS-010759 compared to shC OCI-AML3 (IC50; 8.6±4.2 nM vs 0.5±0.1 nM, respectively), albeit remained in low nM concentrations. Interestingly, baseline level of p-AMPK expression in parental OCI-AML3 cells was markedly suppressed under physiologic hypoxic (1% oxygen) conditions, which caused activation of mTOR signaling (upregulation of p-4EBP1) and decreased sensitivity to IACS-010759 (IC50; 14.5±7.1 nM).Taken together, these results indicate that the responsiveness of AML to IACS-010759 depend at least in part on the AMPK-mTOR signaling, the gauge of cellular energy metabolic capacity. In sensitive AML cells IACS-010759 induces AMPK activation leading to mTOR suppression which results in cell growth inhibition in AML cells. In contrast, in cells with lower levels of AMPK, or in highly proliferating cells (such as stimulated by IL-3), mTOR is constitutively activated conferring resistance to Oxphosi, possibly through glycolytic adaptation. Altogether, these findings highlight AMPK and mTOR as putative biomarkers of anti-leukemia activity of the novel OxPhosi IACS-010759. DisclosuresAndreeff:Daiichi Sankyo: Consultancy.

MAP4K1 functions as a tumor promotor and drug mediator for AML via modulation of DNA damage/repair system and MAPK pathway.

Background: Acute myeloid leukemia (AML) is a group of heterogeneous hematologic malignancies correlates with poor prognosis. It is important to identify biomarkers for effective treatment of AML. Kinases participate in many regulatory pathways and biological activities in AML. Previous studies demonstrated that MAP4K1, a serine/threonine kinase, was associated with immune regulation and cancer progression. However, its role and mechanism in acute myeloid leukemia (AML) have not been explored.Methods: RNA-seq profiling was performed for Homoharringtonine (HHT)-resistant and Homoharringtonine (HHT)-sensitive cell lines. Bioinformatic tools were used for differential analysis. Cell culture and transfection, Cell proliferation, apoptosis and Cell cycle assay, Quantitative RT-PCR, and Western blotting analysis were used to explore biological phenotypes in vitro.Findings: We found that MAP4K1 was highly expressed in HHT-induced resistant AML cell lines. In addition, overexpression of MAP4K1 in AML cells induced resistance of AML cells against HHT. Not only that, the findings of this study showed that overexpression of MAP4K1 was an independent risk factor that predicts poor prognosis of AML. Further, In vitro studies showed that MAP4K1 modulated cell cycle through MAPK and DNA damage/repair pathways. Therefore, MAP4K1 is a potential target for developing therapies for AML.Interpretation: This study demonstrates that MAP4K1 not only regulates HHT resistance but also independently predicts AML prognosis. In addition, understanding the regulatory mechanism of MAP4K1 reveals novel treatment strategies for resistant and refractory AML.Fundings: This work was supported by the National Natural Science Foundation of China (NSFC) (Grant No.81800199, 81670124, 82070118) and the Natural Science Foundation of Zhejiang Province (LY20H080008).

Activity of Carfilzomib (CFZ) in Acute Myeloid Leukemia (AML) As a Single Agent and in Novel Combinations

Background:Recent advances in targeted therapy have expanded the available therapeutic optionsfor patients with AML. However, many patients still have suboptimal outcomes, particularly in the relapsed/refractory setting, underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs), such as bortezomib, exhibit antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis. CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib making it an agent of interest in AML therapy. Here we assessed the activity of CFZ as a single agent and in novel combinations with Ara-C and/or other agents targeting potential vulnerabilities in AML cell lines. Methods:20 AML cell lines were treated with a single dose of CFZ for 7 days, proliferation inhibition was measured using an IC50 cutoff for CFZ of 10 nM. 2 sensitive (ML2 and MV411) and 2 resistant (AML193 and NOMO1) cell lines were selected for further analysis. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM and Ara-C 200 nM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). Cell lines were aligned according to CFZ IC50. Several proteins involved in various physiological pathways exhibited a potential correlation with CFZ sensitivity. Combination treatments with CFZ and agents targeting these pathways were carried out in selected cell lines. Results:Single-agent CFZ induced apoptosis with apoptotic rates &amp;gt;85% in sensitive cell lines and only 10% in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive, but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis as the major mechanism of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ was combined with Ara-C. RPPA revealed that AML cell lines with higher expression of autophagy-related proteins (Atgs) were more resistant to CFZ treatment. Combining autophagy inhibitor hydroxychloroquine (HCQ) or ROC-325 with CFZ produced a synergistic effect to induce apoptosis in several CFZresistant cell lines. RPPA also revealed that lower basal levels of fatty acid synthase (FASN), a key enzyme involved in lipogenesis, correlated with CFZ sensitivity and CFZ resistant lines tendedto have higher basal FASN levels. The combination of CFZ with a FASN inhibitor resulted in a significant synergistic apoptosis-inducing effect that was observed in the AML lines tested. Conclusion:CFZ demonstrated single agent activity in the nanomolar range in human AML cell lines. The addition of standard-of -care chemotherapy to CFZ did not show antagonism. Combining CFZ with agents targeting autophagy or lipid-metabolism showed synergistic effect in apoptosis. These results suggest a role for CFZ in combination therapeutic strategies for AML patients. Disclosures Mcdermott: TORL Biotherapeutics:Current equity holder in private company;1200 Pharma:Current equity holder in private company.Slamon:TORL Biotherapeutics:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;1200 Pharma:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Research Funding;Eli Lilly:Consultancy;Bayer:Consultancy, Research Funding;Pfizer:Consultancy, Other: stock, Research Funding;Syndax:Research Funding;Aileron:Research Funding;Genetech:Research Funding;Biomarin:Membership on an entity's Board of Directors or advisory committees;Seattle Genetics:Other: Stock;Amgen:Other: Stock.Larson:BMS, Bioline, Celgene, Juno, Janssen:Research Funding;TORL Biotherapeutics:Current equity holder in private company.

A Functional Profiling of Microenvironmental Factors and Small Molecules Reveals Monocyte Chemoattractant Protein-1 Mediates Drug Resistance in Acute Myeloid Leukemia

Background: The treatment of acute myeloid leukemia (AML) patients is a formidable challenge, due to disease heterogeneity and the ability to acquire secondary mutations in response to treatment. As a result, persistence and expansion of residual AML clones prevents complete remission. Emerging evidence suggests that extrinsic factors from the microenvironment protect AML cells from therapy, promote disease progression and drug resistance. Previous work from our lab highlights that higher levels of proinflammatory cytokines in the microenvironment, promote AML progression. This emphasizes the rationale to implement targeted therapies that block extrinsic signaling along with tumor intrinsic changes to overcome drug resistance in AML. Methods and Results: To identify factors and pathways that promote drug sensitivity and resistance in AML, we quantified the levels of 41 cytokines and growth factors from the plasma of 350 newly diagnosed AML patient samples using a multiplex luminex assay. The same cohort were analyzed for drug response to 122 small molecule inhibitors using an ex vivo functional drug sensitivity assay. These data were integrated with the gene expression and reactome pathway data to identify differentially regulated pathways and markers for drug response. Data integration revealed that protein synthesis pathways were significantly enriched in venetoclax, (Bcl-2 inhibitor) resistant AML samples, whereas, cytokine and immune pathways such as aberrant activation of monocyte chemoattractant protein-1 (MCP-1; p= 4.9e-07) were correlated with resistance to trametinib, a MEK inhibitor. In contrast, trametinib sensitivity was associated with activation of IL-1 and TLR signaling, suggesting specific extrinsic pathways may drive specific drug response. As a proof-of-concept, we dissected the role of MCP-1 activation in mediating trametinib resistance in AML. For this, we treated primary AML cells with MCP-1 in the presence of trametinib that led to a 4-fold increase in IC50 (0.4 vs 2.4 µM). To model drug response in vitro, we generated trametinib resistant AML cell lines MOLM13 and MV4;11 (FLT3-ITD mutated) and OCI-AML2 (DNMT3A mutated) by culturing cells in trametinib continuously over 4 months. All the trametinib resistant AML cell lines showed 2-10-fold increase in MCP-1 levels in whole cell lysate and in the conditioned media compared to the parental cells. Further, a long-term exposure of AML cells to MCP-1 in the presence of trametinib conferred a growth advantage with a 4-fold increase in cell numbers in comparison to the cells cultured with trametinib alone (n=4/condition). To delineate the molecular pathways driven by MCP-1 in trametinib resistance, we performed global phosphoproteomics using AML cell lines. We identified that MCP-1 activated various pro-survival pathways such as ERK, JNK, SRC, P70, and PKC kinases as well as cell cycle regulatory proteins as early as 5 mins in trametinib resistant cells. Targeting MCP-1 receptor-CCR2 by pharmacological inhibition, significantly reduced viability of trametinib resistant AML cells by 3-fold. The treatment with CCR2 inhibitor or trametinib individually reduced p-JNK levels by 3-fold in AML cells. A combined treatment with CCR2 inhibitor and trametinib, showed a 7-fold reduction in p-JNK levels. These data reinforced the role of MCP-1 in conferring trametinib resistance and that blockade of MCP-1 signaling re-sensitizes AML cells to trametinib. Conclusion: We show that MCP-1 augments trametinib resistance in AML by triggering novel signaling pathways. These effects can be reversed by blockade of MCP-1-CCR2 axis. Such combination treatment strategy incorporating extrinsic pathways together with the intrinsic ones would aid in re-sensitizing AML cells to therapy and represent an attractive new strategy. Our functional screen identifies that these extrinsic signals modulating drug response are unique to specific targeted therapy and should be considered when designing new therapies and clinical trials. We also created a large resource with 350 primary AML samples correlating microenvironment factors and gene expression with response to 122 small molecules, many of those which are FDA approved. This resource will help identify new drug response pathways in the context of microenvironment to design novel treatment regimens for preclinical and clinical testing and to improve AML outcomes. Disclosures Tyner: Syros: Research Funding; Agios: Research Funding; Incyte: Research Funding; Seattle Genetics: Research Funding; Takeda: Research Funding; Petra: Research Funding; Janssen: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Array: Research Funding; AstraZeneca: Research Funding.

Abstract 5942: An integrated approach reveals novel extrinsic pathways and cytokine signatures associated with drug response in acute myeloid leukemia

Abstract Background: Acute myeloid leukemia (AML) is a heterogeneous disease with a dismal 5 year survival rate below 30%. Current AML therapies have provided little improvement in achieving complete remission. This is attributed to the development of drug resistance and extrinsic factors from the microenvironment that promote AML progression. Previously, our lab demonstrated that proinflammatory cytokines from the bone marrow microenvironment such as IL-1, promote AML progression. This emphasizes the rationale to design and implement targeted therapies to block extrinsic pathways coupled with intrinsic pathways conferring drug resistance in AML. Methods: To identify factors that promote drug sensitivity and resistance in AML, we used a cohort of 350 AML patients with various genetic subtypes. We quantified levels of 41 growth factors in plasma samples using a multiplex luminex assay. The same cohort of AML samples were analyzed for drug response to 130 small molecule inhibitors in in vitro functional drug sensitivity assay, for RNA-seq gene expression, and whole exome sequencing. The data was integrated to identify differential pathways and markers for drug response. To model drug response in vitro we created a series of drug resistant AML cell lines by culturing these cells in specific drugs long-term. Results: Pathway analysis integrating drug sensitivity, transcriptome, and cytokine expression data identified distinct differentially regulated pathways for various inhibitors. We found that protein synthesis pathways were significantly enriched in sorafenib (FLT-3 inhibitor) resistant samples whereas cytokine and immune pathways (such as TLR and MCP1 signaling) were correlated with trametinib (MEK inhibitor) and venetoclax (Bcl-2 inhibitor) resistance. Specifically, venetoclax resistance correlated with augmented levels of cytokines such as IL-1 and IP-10 and growth factors such as PDGF. Further, trametinib resistance in AML samples is correlated with the upregulation of CCL11, MCP1, and TGFα but a downregulation of CCL22 levels. Accordingly, using AML cell line models, we observed an increase in MCP1 and TGFα at the transcript and secreted protein levels in trametinib resistant cell lines. We demonstrated that MCP1 stimulation activates survival pathways by activating pERK and pJNK signaling, thus reducing apoptosis in AML cells. Targeting MCP1 in combination with trametinib reverses these effects, thus offering a novel therapeutic approach to overcome drug resistance. Conclusion and perspectives: Our data suggest that distinct extrinsic pathways may regulate the response to specific targeted therapy, thus providing a strong basis to design new treatment regimens. Combination treatment incorporating extrinsic pathways would aid in sensitizing AML cells to therapy. Our study offers an integrated approach and a resource to identify such pathways for a large number of tested drugs, and narrows down functionally important pathways associated with drug response. Citation Format: Rucha V. Modak, Alisa Damnernsawad, Ted Laderas, Guanming Wu, Jeffery W. Tyner, Karin D. Rodland, Shannon K. McWeeney, Anupriya Agarwal. An integrated approach reveals novel extrinsic pathways and cytokine signatures associated with drug response in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5942.

LncRNA MEG3 contributes to drug resistance in acute myeloid leukemia by positively regulating ALG9 through sponging miR-155.

The development of drug resistance is the main obstacle for successful treatment in acute myeloid leukemia (AML). Noncoding RNAs have been implicated in biological function in AML drug resistance. Aberrant protein glycosylation is associated with AML progression. The aim of the study was to explore the potential regulatory mechanism of lncRNA MEG3/miR-155/ALG9 axis in drug resistance of AML. QRT-PCR and Western blot were used for comparison analyses of ALG9, MEG3, and miR-155 levels. CCK-8 and colony formation assays were determined for drug sensitivity and proliferative capability of AML cells. Luciferase reporter assay was used to confirm the targets of miR-155. The mannosyltransferase ALG9 and MEG3 was downregulated in peripheral blood mononuclear cells (PBMCs) of M5/multidrug resistance (MDR) AML patients and adriamycin (ADR)-resistant AML cell lines, which determined a positive correlation in AML patients. Low expression of ALG9 and MEG3 predicted poor prognosis of AML patients. The altered level of ALG9 was found corresponding to the drug-resistant phenotype and sphere formation of AML cells. MiR-155 was overexpressed in M5/MDR patients and ADR-resistant AML cells, as well as inversely correlated to ALG9 expression. MEG3 was a direct target of miR-155 and could sponge miR-155 in AML cells. MEG3 interacted with miR-155 to regulate ALG9 expression, which reversed the effects of ALG9 regulation on proliferation and drug resistance in AML cells. MEG3 sponged miR-155 by competing endogenous RNA (ceRNA) mechanism, which further modulated ALG9 expression and AML procession, providing a novel therapeutic target for AML chemoresistance.

MAP4K1 Functions as a Tumor Promotor and Drug Mediator for AML &lt;i&gt;via&lt;/i&gt; Modulation of DNA Damage/Repair System and MAPK Pathway

Background: Acute myeloid leukemia (AML) is a group of heterogeneous hematologic malignancies with poor prognosis. Finding core biomarkers was an ugent need. Kinases are involved in many regulatory pathways and biological activities in AML. MAP4K1 encodes a serine / threonine protein kinase that is involved in various signaling pathways, however, its role and mechanism in acute myeloid leukemia (AML) has not been explored. Methods: RNA-seq profiling was performed for HHT-resistant and HHT-sensitive cell lines. Bioinformatics were used to make differential analysis. Cell culture and transfection, cell proliferation, apoptosis and cell cycle assay, Quantitative RT-PCR and Western blotting analysis were used to explore biological phenotypes in vitro. Findings: In this study, high expression levels of MAP4K1 were observed in Homoharringtonine (HHT) -induced resistant AML cell lines. In addition, overexpression of MAP4K1 in AML cells induces resistance of AML cells to HHT. Not only that, the findings of this study show that overexpression of MAP4K1 is an independent risk factor for poor prognosis of AML. Further, the results show that expression of MAP4K1 modulates cell cycle through MAPK and DNA damage pathways. Therefore, MAP4K1 is a potential target for development of AML therapy. Interpretation: This study demonstrates that MAP4K1 was not only a mediator of HHT resistance but an independent risk factor for AML prognosis. In addition, information on regulatory mechanism of MAP4K1 provides a novel treatment strategy for resistant and refractory AML. Fundings: This work was supported by National Natural Science Foundation of China (NSFC) (Grant No.81800199, 81670124, 82070118) and the Natural Science Foundation of Zhejiang Province (LY20H080008). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee.

Overcoming Adaptive Therapy Resistance in AML By Targeting Immune Response Pathways

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

CDK8 Inhibitors Induce Transcriptional Reprogramming of AML Cells Associated with Differentiation

Introduction Targeting Cyclin Dependent Kinase 8 (CDK8) is a novel therapeutic strategy for Acute Myeloid Leukemia (AML), a disease associated with poor outcome. CDK8 has been implicated in the regulation of transcription, however antileukemic mechanism of CDK8 inhibitors (CDK8i) are not fully elucidated. SEL120 is an orally available, selective inhibitor of CDK8 and its paralog CDK19. Preclinical characterization of SEL120 indicated a potent inhibition of CDK8, accompanied by transcriptional changes. A first-in-human phase Ib clinical trial with SEL120 in patients with AML or higher-risk myelodysplastic syndrome (HR-MDS) was initiated in June 2019. Inclusion criteria for patients include relapsed or refractory disease and no more than 3 prior lines of standard therapy. To clarify the role of CDK8 in AML, we have used SEL120 and other chemically- unrelated CDK8i to characterize transcriptional and epigenetic changes associated with the antileukemic activity. Material and methods A panel of CDK8i- resistant and sensitive AML cell lines was treated with SEL120 and other CDK8i. Global transcriptional changes were assessed by RNAseq at different timepoints in order to capture early and long-term effects of CDK8i. Transcriptome- wide kinetics of RNA synthesis and turnover in AML cells was captured by Thiol (SH)- Linked Alkylation for the Metabolic sequencing of RNA (SLAMseq). Transcriptional changes and promoter- enhancer interaction analysis data were correlated with chromatin immunoprecipitation results using antibodies against subunits of Polycomb repressive complex 2 (PRC2), trimethylation of histone H3 at lysine 27 (H3K27me3), CDK8 and RNA polymerase II (RNAPII). Results Viability screening with three structurally- unrelated CDK8 inhibitors identified CDK8i- responsive AML cell lines. Transcriptional profiling after CDK8i treatment revealed profound activation of SE- associated genes in both responder and non- responder cell lines, at least partially disconnecting these effects from the anticancer activity of CDK8i. Although SEL120 significantly repressed many genes, including oncogenic MYC-dependent signatures, broad transcriptional profiling indicated rather stimulatory effects on gene expression in AML cells. Detailed transcriptomic analysis of AML cells revealed that SEL120 induced expression of many genes involved in lineage controlling functions. Derepression of genes marked with H3K27me3, including homebox family of transcriptional factors, was one of the earliest transcriptional events in sensitive AML cells treated with SEL120. Morphological changes in AML blasts required prolonged exposure to SEL120 and were correlated with increased expression of lineage markers. A particularly high increase was observed for CD38 and erythroid markers, including erythroid transcription factors - GATA-binding factor 1 and 2 (GATA1/2), and hemoglobin genes. Forced differentiation and reduced viability were also confirmed in patient- derived cells treated with SEL120. Conclusions CDK8 inhibitors act both by inducing expression and repression of distinct genes. Our study identifies that CDK8i cause epigenetic reprogramming that results in antileukemic efficacy by activation of H3K27me3 differentiation programs and inhibition of MYC-dependent genes. Because PRC2- dependent H3K27me3 maintains the stemness by repression of lineage commitment genes, our data suggest that CDK8i may negatively affect core properties of LSCs. Collectively, our studies demonstrated underlying mechanisms of anticancer activity of SEL120 in AML cells, currently investigated in clinical studies. Disclosures Mazan: Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Wiklik:Selvita S.A.: Employment. Combik:Selvita S.A.: Employment. Golas:Selvita S.A.: Employment. Masiejczyk:Selvita S.A.: Employment. Fiedor:Selvita S.A.: Employment. Malusa:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Mikulski:Selvita S.A.: Employment. Chrom:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rzymski:Selvita S.A.: Employment, Equity Ownership.

CircPAN3 contributes to drug resistance in acute myeloid leukemia through regulation of autophagy

The aim of this study was to investigate the role and underlying mechanism of circular RNA (circRNA) circPAN3 in mediating drug resistance in acute myeloid leukemia (AML). We first established two doxorubicin (ADM)-resistant AML cell lines and then utilized high-throughput RNA sequencing (RNA-seq) to compare their circRNA expression profiles with those of the parental cell lines. With bioinformatic analysis, we identified key circRNA molecules involved in drug resistance and validated our findings in clinical specimens. The target microRNAs (miRNAs) and downstream mRNAs were also explored bioinformatically. Using RNA interference technique, the potential mechanism was further investigated. Twenty-nine circRNAs were identified to be differentially expressed between ADM-resistant and sensitive cells. We found that circPAN3 is most likely a key mediator in the development of AML drug resistance, evidenced by the increased expression in ADM-resistant cell lines and BM samples from relapsed patients. Additionally, downregulation of circPAN3 by small interfering RNA (siRNA) significantly restored drug sensitivity to ADM in the two ADM-resistant cell lines, but lentivirus-mediated circPAN3 overexpression had the opposite effect. Subsequent bioinformatic analysis and mechanistic experiments revealed that circPAN3 may facilitate AML drug resistance through regulating autophagy and influencing expression of apoptosis-related proteins, while AMPK/mTOR signaling plays a key role in the regulation of circPAN3 on autophagy. These findings may provide new important insights into the role of circRNAs in mediating AML drug resistance, and suggest that circPAN3 might be a potential target for treatment of drug-resistance AML, which merits further investigation and validation.

Abstract 297: Activity of carfilzomib (CFZ) alone and in combination with cytarabine and chloroquine diphosphate (CHQ) in acute myeloid leukemia (AML)

Abstract Background: Despite the recent advances in AML with the emergence of multiple targeted therapies, patient outcomes remain poor, particularly in the relapsed/refractory setting underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs) have antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis with multiple clinical trials showing promising activity of bortezomib particularly when combined with other cytotoxic agents (Csizmar 2016). CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib and is therefore an agent of interest in AML therapy (van der Helm 2015). Here we assessed the activity of CFZ alone and in combination with cytarabine and the autophagy inhibitor CHQ in AML cell lines. Methods: After screening 18 AML cell lines, 3 sensitive: KASUMI1, ML2, and MV411, and 3 resistant cell lines: AML193, THP1, and NOMO1, were selected for further analysis using an IC50 cutoff for CFZ of 10 nM. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM, cytarabine 200 nM, and CHQ 20 µM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA) and confirmed with western blot analysis. Results: Single-agent CFZ induced apoptosis in sensitive cell lines with apoptotic rates increasing by 85% to 95%; while apoptotic rates remained unchanged in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis and cell cycle arrest as the major mechanisms of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ 10 nM was combined with cytarabine (200 nM). RPPA revealed that AML cell lines with low expression of autophagy related proteins are more sensitive to proteasome inhibition with CFZ treatment compared to resistant cell lines. This was confirmed with western blot which showed upregulation of the autophagy related proteins in resistant cell lines upon exposure to CFZ. However, sensitive cell lines did not upregulate autophagy pathways which may lead to cell cycle arrest and apoptosis. Inhibiting autophagy with CHQ sensitized CFZ resistant lines to CFZ treatment. Conclusion: CFZ demonstrated single agent activity in human AML cell lines and upregulation of autophagy may contribute to resistance to CFZ which can be overcome using autophagy inhibitors such as CHQ. These results support the development of CFZ and autophagy inhibitor-based combinations for AML. Citation Format: Mao Yu Peng, Yasmin Abaza, Dylan Conklin, Erika Von Euw, Monica Mead, John Timmerman, Dennis Slamon, Sarah Larson. Activity of carfilzomib (CFZ) alone and in combination with cytarabine and chloroquine diphosphate (CHQ) in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 297.

Global Gene Expression Analysis Reveals Genotoxic Effect of Nrf2 Pharmacological Inhibitor Brusatol in Myeloid Leukemia Cells- a Promising Novel Anticancer Agent

Combating drug resistance is a paramount task in treating acute myeloid leukemia (AML). NFE2 related factor 2 (NRF2) is a major player in inducing chemo resistance in both AML and solid tumors. We have previously reported that inhibition of NRF2 both by shRNA knockdown and pharmacological inhibition using brusatol sensitizes inherent resistant AML cell lines and patient samples to chemotherapy (Karathedath et al, Plos one, 2017). Recent studies have shown that brusatol acts as a potent protein translation inhibitor in A549 lung cancer cell line and this deleterious effect is due to the molar scale concentrations of the drug (Stokoe, et al., 2016, Harder et al, 2016).As the available reports was in solid tumor lines and at higher concentrations, we undertook this study to elucidate the mechanism of action of brusatol in our experimental setup utilizing Nano molar concentration of brusatol in AML cell lines. We compared the global gene expression changes induced by brusatol in AML cell lines resistant to Ara-C, Dnr and ATO namely THP-1 and U937.Total RNA was extracted from both cell lines with and without brusatol treatment (100nM for 6 hours) using Qiagen RNeasy mini kit, cDNA was synthesized and subjected to a One -color 8X60K Agilent micro-array analysis. Data normalization, and analysis was performed using Gene Spring GX (V 12.0) software using the 75th percentile shift. Using student t-Test, 3070 genes were identified to be differentially expressed between THP-1 control and brusatol treated while 6856 genes were identified to be differentially expressed between U937 control and brusatol treated samples. About 1242 differentially expressed genes were common between brusatol treated THP-1 and U937. Differentially expressed genes(DEGs) were clustered using hierarchical clustering based on Pearson coefficient correlation algorithm to identify significant gene expression patterns. Genes were classified based on functional category using biological analysis tool DAVID. The enriched genes in both THP-1 and U937 after brusatol treatment fell into the following; Molecular function: protein binding (1396 and 1701 genes), Biological function: regulation of transcription (350 and 502 genes), Cellular component: Mitochondrion (231 and 249 genes) and so on. KEGG analysis was performed to identify enriched pathways after brusatol treatment in DEGs. DEGs in THP1 and U937 were enriched in 33 and 57 pathways were respectively, combining the pathways revealed 20 common target pathways between THP1 and U937. Pathways involved in cellular stress response such as NFkB, TNF and MAPK pathways were selectively up-regulated in the common target pathways and genes involved in cell cycle pathway were down regulated. Select genes from these target pathways and few genes DOCK3, KLF10, NR4A3, GADD45B, KDM4B, GPR155, H2FAY, from the top 100 DEGs were validated by RQ-PCR (SyBr green) all the genes screened showed results consistent with the micro-array data.Interestingly DNA damage response genes GADD45B, KDM4B, H2AFY were in the top up-regulated genes indicating that brusatol might also cause DNA damage in leukemic cells. NR4A3 the orphan nuclear receptor is an established tumor suppressor in AML and is generally silenced in both AML cell lines and primary samples. One of the key finding from our data is that brusatol strikingly up-regulated NRA4A3 expression in both THP1 and U937. This study has shed light into other modes of action of brusatol apart from being a modulator of the protein translational machinery. Mechanism of chemo sensitization by brusatol could be tissue, time and concentration specific. Our data reveals brusatol at nano-molar concentration acts as a potent DNA damaging agent and reactivates NR4A3 expression in AML apart from being an inhibitor of NRF2. We have also reported at nano-molar concentration brusatol has minimal toxicity towards normal Peripheral blood mono-nuclear cells. Brusatol could be an ideal compound to combat drug resistance in AML. Further experimental validation is warranted to substantiate these finding of the mode of action of brusatol in chemo sensitizing AML cells. DisclosuresNo relevant conflicts of interest to declare.

Combined Targeting of BET Family Proteins and BCL2 Is Synergistic in Acute Myeloid Leukemia Cells Overexpressing S100A8 and S100A9

BackgroundThe 5-year survival rate for acute myeloid leukemia (AML) remains poor with most patients succumbing to relapse or refractory disease. Recently, the BCL2 specific inhibitor venetoclax has shown promising anti-leukemia activity in high-risk AML patients. Most patients, however, ultimately develop resistance to monotherapy and novel combination treatments with venetoclax are needed for patients with no other therapy options available. In this study we identified high expression of calcium binding protein S100A8/S100A9 to be associated with venetoclax resistance and looked for drug combinations to overcome the resistance in AML patient samples overexpressing S100A8 and S100A9.MethodsGene expression was assessed by RNA sequencing of AML patient samples and validated by qPCR. Gene enrichment analysis was performed on differentially expressed genes between venetoclax highly sensitive (n=3) and resistant (n=4) samples. Sensitivity of AML patient derived mononuclear cells was assessed to 304 different small molecule inhibitors by measuring cell viability after 72 h incubation with 5 different concentrations (1-10,000 nM) using the CellTiter-Glo (CTG) assay. A drug sensitivity score (DSS) was calculated based on a modified area under the dose response curve calculation. Drug combination studies were performed using AML cell lines resistant to venetoclax and confirmed with primary patient cells (n=15). Data of the drug combination studies were analyzed with the Zero Interaction Potency (ZIP) model by considering a dose-response matrix where two drugs are tested at 8 concentrations in a serially diluted manner. Statistical dependence between gene expression and drug sensitivity data was assessed by Pearson's correlation coefficient modelling.ResultsVenetoclax resistant AML patient samples were found to overexpress genes related to immune responses including inflammatory related proteins S100A8 and S100A9. The expression of S100A8 and S100A9 was upregulated in a sub-group of AML patients with somatic mutations in DNA methylation genes IDH2 and TET2 and chromatin modifier ASXL1. Functional studies with AML cell lines validated high expression of the S100 proteins in cells insensitive to venetoclax (NOMO-1, SKM-1 and SHI-1) whereas sensitive cell lines (MOLM-13, Kasumi-1 and ML-2) did not express the proteins. Integrated analysis of S100A8 and S100A9 expression and ex vivo drug sensitivity data indicated positive correlation of S100 expression with sensitivity to BET inhibitor (birabresib), PI3K inhibitor (TG100-115) and MEK1/2 inhibitor (AZD8330). In contrast, sensitivity to venetoclax and the FLT3 inhibitor quizartinib negatively correlated with S100 gene expression. Subsequently, we combined positively correlating drugs with venetoclax and tested the efficacy of these combinations in AML cell lines and patient samples. From the drug combination studies we found that BET inhibitor birabresib was able to overcome resistance to venetoclax treatment. The BCL2/BET inhibitor combination was synergistic in venetoclax resistant cell lines NOMO-1 and SKM-1, which express high-levels of S100A8 and S100A9 (Figure 1A). Efficacy of the combination on primary AML patient samples correlated with the expression level of the S100 genes. Nine of 11 high expression samples were sensitive to the venetoclax/birabresib combination (Figure 1B-C), whereas no synergy was observed in 3 of 4 samples with a low level of S100 expression.ConclusionsThe calcium binding proteins S100A8 and S100A9 are abundant in myeloid cells and are associated with poor prognosis in AML (Edgeworth et al, J Biol Chem. 1991; Nicolas et al, Leukemia 2011). From ex vivo and in vitro analyses of AML, we found that high expression of S100A8 and S100A9 is highly correlative with resistance to the BCL2 inhibitor venetoclax. In contrast, high S100A8 and S100A9 expression correlates with sensitivity to BET inhibitor birabresib. Interestingly, our studies showed that these two drugs act synergistically in venetoclax resistant AML cell lines and AML patient samples and may be a beneficial novel combination that should be further confirmed in preclinical and clinical investigations. [Display omitted] DisclosuresPorkka:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Heckman:Orion Pharma: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

Role of NF-E2 related factor 2 (Nrf2) on chemotherapy resistance in acute myeloid leukemia (AML) and the effect of pharmacological inhibition of Nrf2.

Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell’s ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.