Promising Therapeutic Target In Acute Myeloid Leukemia Research Articles

LILRB4 in acute myeloid leukemia: From prognostic biomarker to immunotherapeutic target.

Leukocyte immunoglobulin-like receptor (LILR) B4 (also known as ILT3/CD85k) is an immune checkpoint protein that is highly expressed in solid tumors and hematological malignancies and plays a significant role in the pathophysiology of cancer. LILRB4 is highly expressed in acute myeloid leukemia (AML), and this phenotype is associated with adverse patient outcomes. Its differential expression in tumors compared to normal tissues, its presence in tumor stem cells, and its multifaceted roles in tumorigenesis position it as a promising therapeutic target in AML. Currently, several immunotherapies targeting LILRB4 are undergoing clinical trials. This review summarizes advancements made in the study of LILRB4 in AML, focusing on its structure, ligands, expression, and significance in normal tissues and AML; its protumorigenic effects and mechanisms in AML; and the application of LILRB4-targeted therapies in AML. These insights highlight the potential advantages of LILRB4 as an immunotherapeutic target in the context of AML.

GADD45a Controls Self-Renewal in Acute Myeloid Leukemia Stem Cells

Acute myeloid leukemia (AML) is a heterogenous malignancy, where the persistence of chemo-resistant leukemia stem cells (LSCs) contributes to disease relapse. We have previously demonstrated the clinical significance of WNT/β-catenin signaling in driving AML LSCs (Science, 327:1650-1653, 2010; Cancer Cell, 38:1-16, 2020). In this study, we uncover that GADD45a (growth arrest and DNA-damage inducible protein) is an essential regulator of β-catenin signaling pathway and its loss promotes LSC function and leukemia progression. Transgenic knockout of Gadd45a led to a progressive increase in aberrant self-renewal and leukemogenesis in vivo. Gadd45a-/- leukemic cells developed a more aggressive leukemia with a shorter latency than Gadd45a+/+ cells in mice, indicating the involvement of Gadd45a loss in AML initiation and progression. Subsequent serial transplantation experiments showed that Gadd45a deletion enhanced LSC self-renewal in vivo. In agreement with our findings in murine LSCs, deletion of GADD45a by CRISPR/Cas9 in AML patient-derived xenograft (PDX) cells revealed increased engraftment and tumor burden in NSG mice. Consistent with our phenotypic observations, knockout of GADD45a increased βcatenin activity and key WNT/self-renewal target genes in human AML cells. In addition, our cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) data showed that GADD45a deletion in patient-derived LSCs was associated with cell metabolism, reactive oxygen species and tumor progression, as well as poor patient outcomes in AML. Further studies are being conducted to evaluate transcriptional mechanisms discovered by our single-cell sequencing. Taken together, this study is the first to demonstrate that GADD45a loss promotes LSC potential and consequently enhances tumor growth in murine and PDX models of AML, thus showcasing GADD45a as a promising therapeutic target in AML.

Side-by-side comparison of BH3-mimetics identifies MCL-1 as a key therapeutic target in AML

Despite advances in the treatment of acute myeloid leukemia (AML), prognosis of AML patients is still dismal and better treatment options are required. B-cell Lymphoma 2 (BCL-2) homology domain 3 (BH3)-mimetics are emerging as a novel class of apoptosis-inducing agents that are currently being tested for the treatment of different hematological malignancies including AML. Particularly, the selective BCL-2 inhibitor ABT-199/Venetoclax is demonstrating clinical responses and has recently been approved in combination for the treatment of AML. Compounds targeting the related protein MCL-1 have recently entered clinical trials, highlighting the urgency to compare the different BH3-mimetics and identify the most promising antiapoptotic target in AML. We performed a side-by-side comparison of different highly selective and potent BH3-mimetics targeting BCL-2 (ABT-199), MCL-1 (S63845) or BCL-xL (A1331852) in a panel of AML cell lines and primary patient cells. Gene knockdown using siRNAs was utilized to investigate the functional relevance of BCL-2 proteins. Western blotting and immunoprecipitations were used to explore the influence of BH3-mimetics on interactions between pro- and antiapoptotic BCL-2 proteins. A1331852 induced apoptosis only in selected cases, indicating that BCL-xL is not a very promising therapeutic target in AML. However, S63845 displayed higher potency than ABT-199, with more cell lines and primary cells responding to S63845 than to ABT-199. MCL-1 dependency in AML cells was confirmed by siRNA-mediated knockdown of MCL-1, which was sufficient to induce apoptosis. S63845-induced cell death was accompanied by a displacement of the BH3-only protein BIM as well as BAK, resulting in BAK-dependent apoptosis. In contrast, ABT-199-induced cell death was mediated by BAX rather than BAK, indicating distinct non-redundant molecular functions of BCL-2 and MCL-1 in AML. Our study reveals that MCL-1 may be a more prevalent therapeutic target than BCL-2 in AML and identifies BIM and BAK as important mediators of S63845-induced apoptosis in AML.

Abstract 1297: Casein kinase II (CK2) as a potential therapeutic target in acute myeloid leukemia (AML)

Abstract Casein Kinase II (CK2) is a pro-oncogenic kinase that is overexpressed in a majority of human malignancies, including leukemia. One third of AML cases without detectable high risk molecular alteration have high expression of CK2 which is strongly associated with poor outcome. Previously published data showed that CK2 directly phosphorylates several transcription factors including Ikaros (encoded by IKZF1) resulting in reduced DNA-binding affinity and loss of Ikaros function as a tumor suppressor. Here we present the evidence demonstrating role of Ikaros and CK2 in regulation of apoptosis in AML. We show that CK2 inhibitor, CX4945 has strong anti-leukemic effect in AML and one of the novel mechanisms of action involves restoration of Ikaros function as a transcriptional regulator of genes involved in apoptosis. Results: Treatment of AML cell lines and primary cells with CK2 inhibitor, CX4945 show cytotoxicity (IC50 3-5uM), increased apoptosis, cell cycle arrest and poor colony formation. Treatment of murine xenografts of AML with CX4945 showed significant anti-tumor effect. Further studies dissecting the mechanism of action of CX4945 are as follows: Genome-wide binding studies using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) demonstrated that treatment of AML cell line, U937 with CX4945 at IC50 concentration enhances binding affinity of Ikaros transcription factor at the promoter regions of several target genes. Among the significantly affected genes (more than two fold change) are anti-apoptotic genes such as BCL2A1 (B Cell Lymphoma 2 related protein A1) and Bcl-xL (B Cell Lymphoma extra-Large). Dysregulation of apoptotic pathway is hallmark of cancer cells. These findings were confirmed qChIP where CX4945 treated AML cells showed increased binding of Ikaros to the BCL2A1 and Bcl-xL promoters. We used gain-of-function and loss-of-function experiments to determine how Ikaros regulates BCL2A1 and Bcl-xL genes in AML. Ikaros overexpression using lentiviral transduction results in reduced expression of BCL2A1 and Bcl-xL both at mRNA and protein level. Similar results were seen with silencing of CK2 using CK2 SiRNA as well as CX4945. Increased expression of BCL2A1 and Bcl-xL was noted after Ikaros silencing using SiRNA which did not revert with CX4945 treatment.In conclusion, (1) Ikaros represses transcription of anti-apoptotic genes BCL2A1and Bcl-xL, (2) CK2 directly phosphorylates tumor suppressor Ikaros and inhibits its function as transcriptional regulator, (3) Inhibition of CK2 enhances Ikaros-mediated repression of BCL2A1 and Bcl-xL genes resulting in increased apoptosis, (4) CX4945 has strong anti-leukemia effect in AML xenograft models. CK2 is a promising therapeutic target in AML. Our results unveal a novel mechanism of action of CK2 inhibitor, CX4945 in AML which involves restoration of Ikaros mediated regulation of apoptosis. Citation Format: Badhauria Preeti, Soumya Iyer, Elanora Dovat, Pavan Kumar Dhanyam Raju, Jonathan Payne, Chunhua Song, Yali Ding, Claxton David, Sharma Arati, Chandrika Gowda. Casein kinase II (CK2) as a potential therapeutic target 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 1297.

The kinases IKBKE and TBK1 regulate MYC-dependent survival pathways through YB-1 in AML and are targets for therapy

AbstractTo identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.

CD97 Is a Critical Regulator of Acute Myeloid Leukemia Stem Cell Function

Despite recent advances in our understanding of the genetic origins of acute myeloid leukemia (AML), clinical outcomes remain poor. While standard induction chemotherapy induces remission in most patients, the majority of patients eventually relapses and dies from progressive disease. A number of cell surface proteins have been shown to be expressed at high levels on AML stem cells compared to normal HSCs including CD47, CD44, CD96, TIM3, CD123, CD25 and IL1RAP. Despite the attention these antigens have received, data supporting their roles as cell-intrinsic regulators of LSCs are more limited. We have identified CD97, a member of the adhesion class of GPCR, as a frequently upregulated antigen expressed on AML blasts that is a critical regulator of LSC function.In order to determine whether CD97 is expressed on LSCs, we assessed CD97 expression in human AML primary samples by flow cytometry. This study revealed that CD97 was 10 fold increased on LSC-enriched (CD34+CD38-) blasts compared to cord blood HSCs in all 30 patients tested. In contrast, the expression of other previously reported LSC was more variable and less frequently increased over HSCs. To test if CD97 may regulate leukemic cell function, we knocked-down (KD) CD97 in human AML cell lines. CD97-KD in HL60 and MOLM-13 AML cell lines increased apoptosis, reduced cell growth 4-fold, and delayed mortality once injected into NSG mice (20 vs. 28 days, p=0.0012). As CD97 expression increases during normal myelomonocytic differentiation, we cultured control and CD97-KD HL60 or U937 cells (which can be induced to differentiate along the myelomonocytic lineage following all-trans-retinoic acid (ATRA) treatment) for 4 days in the presence of 10nM ATRA or vehicle control. Flow cytometric analysis revealed that CD97-KD cells expressed higher levels of the myeloid differentiation antigens CD11b and CD15 both before and after ATRA treatment. In addition, mRNA expression of CD11b and PU.1, key genes associated with myeloid differentiation, were increased in CD97-KD cells compared to controls.To investigate whether CD97 regulates leukemic initiation, purified HSPCs from WT and CD97-/- mice were transduced with the MLL-AF9 oncogene (MA9). CD97-/--MA9 cells (GFP+) showed a 3-fold reduction in colony formation in serial replating assays in methylcellulose, consistent with a reduction in self-renewal. Mice transplanted with 5,000 CD97-/--MA9 blasts survived significantly longer than those receiving WT-MA9 blasts (70 vs 100 days, p=0.0039). Consistent with reduced disease aggressiveness, at the time of sacrifice mice injected with WT-MA9 cells contained a higher percentage of total GFP+ cells, L-GMPs and CD11b+Gr1+ cells in the BM than those receiving CD97-/--MA9 grafts. Secondary transplant experiments revealed that CD97-/--MA9 blasts exhibit a 25-fold lower LIC frequency than WT-MA9 blasts. Similar results were obtained using the AML1-ETO9a mice model.To better understand the molecular pathways underlying CD97 regulation of AML cells, we performed a combination of RNA-sequencing and protein phospho-array analyses in CD97-KD and scramble control cells. Gene Ontology and GSEA analyses revealed enrichment of transcripts encoding genes that regulate apoptosis as well as downregulation of cell cycle regulators in CD97-KD cells. Consistent with our flow cytometry data, CD97-KD induced differentiation, as evidenced by increased expression of myeloid differentiation genes and global increases in granulocyte and monocyte fingerprint signatures. Phosphoproteome analysis revealed downregulation of several mediators of the MAPK cascade and MTOR signaling pathways in CD97-KD cells. These signaling changes were confirmed by western blot, as activating phosphorylation events of ERK 1/2, c-Jun, and STAT3 were decreased upon CD97-KD. Together, these data indicate that functional alterations in CD97-KD AML cells are due to the modulation of signaling cascades including MAPK and mTOR.Our studies indicate that CD97 is expressed at a higher frequency in AML patient samples than previously identified LSC antigens and that its expression is elevated, irrespective of genetic subtype. Studies using human cell lines and mouse models of AML demonstrate that CD97 regulates the proliferation and survival of leukemic blasts as well as disease initiation and self-renewal. Collectively, these data credential CD97 as a promising therapeutic target in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Over-expression of FoxM1 is associated with adverse prognosis and FLT3-ITD in acute myeloid leukemia

Forkhead box M1 (FoxM1) drives cell cycle progression and the prevention of growth arrest and is over-expressed in many human malignancies. However, the characteristics of FoxM1 in acute myeloid leukemia (AML) are not clearly understood. We investigated the expression level of FoxM1 and analyzed the correlation of FoxM1 expression with AML patient characteristics and prognoses. Changes in FoxM1 expression were detected after MV4–11 cells, which have an internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 gene (FLT3-ITD), and control THP1 cells (encoding wild-type FLT3) were treated with the FLT3 receptor tyrosine kinase inhibitor AC220 (quizartinib) or FLT3 ligand (FL). Finally, we determined the apoptosis rates after the addition of the FoxM1 inhibitor thiostrepton (TST) to AML cells with or without FLT3-ITD. The expression of FoxM1 in AML patients was correlated with the presence of FLT3-ITD, genetic groups, and possibly overall survival. Inhibition of FLT3-ITD by AC220 down-regulated FoxM1 expression in MV4–11 cells, and stimulation of FLT3 by FL up-regulated FoxM1 expression in MV4–11 and THP1 cells. TST induced the apoptosis of MV4–11 and THP1 cells in a dose-dependent manner. Thus, FoxM1 is a potential prognostic marker and a promising therapeutic target in AML.

The Pim Kinase Inhibitor SGI-1776 Chemosensitizes Multidrug Resistant Cells by Both Inhibiting Drug Transport by ABCB1 and ABCG2 and Decreasing ABCB1 and ABCG2 Surface Expression On Cells That Overexpress Pim-1.

AbstractAbstract 2462Overexpression of the ATP-binding cassette (ABC) cellular drug efflux proteins ABCB1 and ABCG2 on acute myeloid leukemia (AML) cells is associated with inferior chemotherapy outcomes. Nevertheless, inhibitors of drug transport have not improved treatment outcomes in clinical trials. The serine/threonine kinase Pim-1, encoded by a proto-oncogene originally identified as the proviral integration site in Moloney murine leukemia virus lymphomagenesis, is expressed in AML and is implicated in regulation of multiple key cellular processes, as well as drug resistance. Our group has shown that Pim-1 phosphorylates ABCB1 and ABCG2 and promotes their translocation to the cell surface, where they mediate drug efflux. The imidazo[1,2-b]pyridazine small molecule SGI-1776 (Tolero Pharmaceuticals, Inc. Salt Lake City, UT) is the first Pim kinase inhibitor to have entered clinical testing. SGI-1776 has been shown to sensitize ABCB1-overexpressing drug-resistant cells to ABCB1 substrate cancer chemotherapy drugs, but chemosensitization was found to be associated with direct inhibition of drug transport mediated by ABCB1. Moreover, while silencing of Pim-1 expression with siRNA was found to sensitize ABCG2-overexpressing cells to ABCG2 substrate chemotherapy drugs, the effects of SGI-1776 on resistance mediated by ABCG2 have not been studied. Therefore we studied the Pim-1-dependent and -independent effects of SGI-1776 on chemosensitivity of cells overexpressing ABCB1 and ABCG2.SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1 μM decreased the IC50s of ABCB1 and ABCG2 substrate drugs including daunorubicin and mitoxantrone 2- to 4-fold in leukemia and myeloma cell lines overexpressing ABCB1 and ABCG2, but had no effect on the IC50 of the non-substrate drug cytarabine, and no effect in parental cells. SGI-1776 also increased apoptosis of ABCB1- and ABCG2-overexpressing leukemia and myeloma cells exposed to ABCB1 and ABCG2 substrate chemotherapy drugs, respectively, and decreased their colony formation in the presence of substrate, but not non-substrate, chemotherapy drugs, with no effect on parental cells. We found that SGI-1776 decreased ABCB1 and ABCG2 surface expression, measured by flow cytometry, on K562/ABCB1 (p=0.013) and K562/ABCG2 (p=0.0038) leukemia cells, respectively, both of which express Pim-1 at high levels, without decrease in total cellular ABCB1 and ABCG2 expression, measured by Western blot analysis. In contrast, SGI-1776 had no effect on ABCB1 and ABCG2 surface expression on HL60/VCR leukemia and 8226/MR20 myeloma cells, which express ABCB1 and ABCG2, respectively, but express Pim-1 at lower levels. Thus SGI-1776 decreased ABCB1 and ABCG2 surface expression on cells that overexpress Pim-1, consistent with decreased cell surface translocation of ABCB1 and ABCG2 as a result of inhibition of Pim-1, but also chemosensitized cells expressing ABCB1 and ABCG2 in the absence of effects on ABCB1 and ABCG2 cell surface expression. We found that SGI-1776 indeed inhibited uptake of fluorescent substrates of both ABCB1 and ABCG2, measured by flow cytometry, in a concentration-dependent manner. We further determined that SGI-1776 inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [125I]-IAAP and stimulated ABCB1 and ABCG2 ATPase activity, consistent with binding to drug-binding sites of ABCB1 and ABCG2 and inhibition of substrate transport by both proteins. Thus SGI-1776 both inhibits drug transport by ABCB1 and ABCG2 and decreases ABCB1 and ABCG2 surface expression on cells that overexpress Pim-1.Pim-1 is thought to be a clinically promising therapeutic target in AML and other malignancies, and other Pim kinase inhibitors are in preclinical and clinical development. Subsequent clinically applicable Pim kinase inhibitors should be characterized with regard to interactions with ABCB1 and ABCG2. In particular, while therapeutic strategies based on inhibition of drug transport mediated by ABCB1 with competitive inhibitors including PSC-833, zosuquidar and cyclosporin A have largely been clinically unsuccessful, inhibition of ABCB1 and ABCG2 cell surface translocation by Pim kinase inhibitors may have therapeutic implications. Disclosures:No relevant conflicts of interest to declare.