Acute Myeloid Leukemia Leukemic Cells Research Articles

Venetoclax Synergizes with IMGN632, a Novel CD123-Targeting Antibody Conjugated to a DNA Alkylating Payload, By Suppressing DNA Damage Response and Potentiating Apoptosis in Acute Myeloid Leukemia in Vitro Models

CD123, a component of interleukin-3 receptor, is overexpressed on the surface of acute myeloid leukemia (AML) blasts and leukemic stem cells compared with normal hematopoietic progenitors, which makes this antigen an attractive therapeutic target. IMGN632 is a novel antibody-drug-conjugate comprising a high affinity CD123-targeting antibody and DNA alkylating cytotoxic payload. In preclinical studies, IMGN632 demonstrated high potency and selective cytotoxicity against AML leukemic cells vs normal myeloid progenitors, suggesting reduced likelihood of myelosuppression (Kovtun, 2018). Importantly, we previously showed high synergy of IMGN632 combination with BCL-2 inhibitor venetoclax (VEN) and DNA hypomethylating azacytidine (AZA) in AML cell lines and xenograft models (ASH 2020, #617). IMGN632 demonstrated encouraging single agent activity and favorable tolerability in relapsed/refractory AML patients (ASH 2019 #734, ASCO 2020 TPS7563, NCT03386513) and is currently tested in combination with VEN and/ or AZA in relapsed and frontline CD123-positive AML patients (NCT04086264). In this study, we provide new insight into molecular mechanisms underlying the synergistic efficacy of these combinations using AML models in vitro. AML cells with FLT3-ITD mutations expressed higher levels of CD123, as previously reported (Han 2013). FLT3-ITD cell lines were particularly sensitive to IMGN632 with IC 50 values at low ng/ml range. Correspondingly, as evidenced by large scale drug combination screen using BLISS independence model, triple IMGN632/VEN/AZA treatment was synergistic at multiple dosage levels and ratios, suggesting a favorable potency of triple combination in FLT3-mutated subtypes of AML associated with poor prognosis. The efficacy of IMGN632/VEN/AZA combination was however decreased in cells lacking functional TP53. shRNA-mediated knockdown of TP53 in FLT3-ITD MOLM13 cells resulted in increased cell viability, reduced apoptosis and downregulation of cleaved caspase-3 and PARP. While both treated wild-type and TP53 KD cells showed robust overexpression of phosphorylated histone H2AX (p-H2AX), TP53 KD cells expressed higher levels of anti-apoptotic MCL-1 and lower levels of pro-apoptotic BAX, indicative of their intrinsic inability to fully execute apoptosis and resulting in suppressed response to triple combination. Importantly, in sensitive AML cells, IMGN632 induced profound DNA damage, G 1 (MOLM13 and MOLM14 cells) or G 2/M (MV4-11 cells) phase cell cycle arrest and decline in S-phase population, all above augmented by VEN/AZA co-treatment. Observed increase in p-H2AX was accompanied by apoptotic caspase-3 and PARP cleavage and was fully prevented by QVD-OPh caspase inhibitor, suggesting that double-stranded DNA damage and p-H2AX triggered by triple combination were secondary events associated with apoptosis but not the initial cause of cell cycle arrest. Thus, given that cytotoxic payload of IMGN632 is a DNA alkylator, we next tested its impact on mediators of the DNA damage response (DDR). IMGN632 alone induced phosphorylation of p53 at Ser15, a step required for p53 transcriptional activity, and triggered activating phosphorylation of checkpoint kinase Chk1 (Ser345), known to facilitate cell cycle arrest and DNA damage repair, thus preventing treated cells from progressing through S-phase but also increasing the potential of repairing IMGN632-induced DNA damage (Fig. 1A). Upon combined IMGN632+VEN treatment, p53 phosphorylation was sustained, but p-ChK1 decreased, suggesting that VEN may impair protective DDR triggered by IMGN632, resulting in greater cell death. To support this hypothesis, we showed that IMGN632 treatment prior to VEN produced significant synergistic activity (Fig. 1B). Together, these results suggest that VEN, apart from its canonical inhibitory effect on anti-apoptotic BCL-2, exerts previously unrecognized ability to suppress DDR program in AML and augments activity of DNA damaging IMGN632. Failure of cells to sustain DDR in the presence of VEN constitutes a key aspect of high efficacy of IMGN/VEN/AZA combination in AML.

Phase 1/2a Trial of Bomedemstat with or without All- Trans Retinoic Acid (ATRA) in Advanced Myeloid Malignancies

Background: Acute myeloid leukemia (AML) is a devastating disease difficult to treat. Although > 60% of patients (pts) with AML treated with standard therapy achieve complete remission, at least 70% relapse. Overexpression of lysine-specific demethylase 1 (LSD1) mRNA and excess LSD1 protein have been observed in several cancer types, including progenitor populations of leukemic cells in AML. In preclinical studies, LSD1 inhibition impaired tumor growth and induced apoptosis of leukemic cells (Harris et al., 2012). In combination with ATRA, LSD1 inhibition enhanced survival in mouse models of AML (Shenck et al., 2014). In this first-in-human trial, we examine the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of bomedemstat with or without ATRA in pts with high-risk AML and myelodysplastic syndrome (MDS). Methods: In this multicenter, open-label phase 1/2a trial, eligible pts were aged ≥18 years old, with high-risk AML or MDS, ECOG PS ≤2 (AML), and International Prognostic Score (IPSS) of at least intermediate-2 or Revised International Prognostic Score (IPSS-R) of at least intermediate (MDS). Pts could not have received immunotherapy within <8 weeks, chemotherapy within <3 weeks (except hydroxyurea), and radiation to >30% of marrow within <2 weeks of treatment. In the dose-finding phase, pts received bomedemstat at a starting dose of 0.75, 1.5, 3.0, or 6.0 mg/kg once-daily in 14-day cycles (7 days on, 7 days off), either alone or in combination with 45 mg/m 2ATRA for up to 4 cycles. Treatment was allowed to continue upon evidence of clinical benefit. In the duration-finding phase, pts received 6.0 mg/kg bomedemstat with ATRA in 21-day (14 days on, 7 days off) or 28-day (21 days on, 7 days off) cycles. Treatment continued until progression, unacceptable toxicity, or withdrawal. Primary endpoint was safety and tolerability. Secondary endpoints included PK parameters and PD effect on hematopoiesis. Clinical responses were documented per revised/modified Cheson criteria. Data cut-off date was December 13, 2018. Results: A total of 45 pts enrolled with a median age 73 years (range, 42 to 88); 19 (42%) were female. A total of 39 pts had AML with 27 (69%) having an ECOG PS score ≤1. Six pts had MDS with 2 (33%) pts having an IPSS of Very High Risk and IPSS-R of High Risk. A total of 33 pts were enrolled in the dose-finding phase (0.75 mg/kg/d bomedemstat [n=3], 1.5 mg/kg/d [n=6], 3.0 mg/kg/d [n=4], 6.0 mg/kg [n=6], 3.0 mg/kg + ATRA [n=5], 6.0 mg/kg + ATRA [n=9]). Twelve pts enrolled in the duration finding phase, 4 received bomedemstat plus ATRA for 14/28 days, and 8 received bomedemstat plus ATRA for 21/28 days. A total of 21 (47%) pts discontinued, 8 (18%) due to death, 5 (11%) due to withdrawal and 4 (9%) due to physician decision. Any grade adverse events (AE) related to bomedemstat occurred in 42 (93%) pts. The most common were diarrhea (42%), nausea (42%), and thrombocytopenia (38%). Grade ≥3 bomedemstat-related AEs occurred in 30 (66.7%) pts. One pt receiving 6.0 m/kg plus ATRA had a grade 5 AE related to bomedemstat due to respiratory failure. No patient experienced dose-limiting toxicity and no maximum tolerated dose was determined. The table summarizes PK parameters in whole plasma for the dose-finding phase. Based on plasma exposures, the Data Safety Monitoring Committee designated 6.0 mg/kg bomedemstat as the PD dose. Mean percent change (standard deviation) in blast counts was 368% (400) for pts receiving 0.75 mg/kg bomedemstat, 2478% (3279) for 1.5 mg/kg, 10% (25) for 3 mg/kg, 2841% (6047) for 6 mg/kg/d. For pts with AML, overall median EFS was 0.97 months (mo) (95% CI, 0.93-1.46), and median OS was 18.8 mo (95% CI, 3.86 to NR). The 6-mo rate for EFS and OS were 9% (95% CI, 0-46%) and 64% (95% CI, 25%-86%), respectively. The overall response rate was 28.2% (95% CI, 15.0-44.9, [1 PR, 10 SD]). For pts with MDS, median EFS was 0.93 mo (95% CI, 0.57 to NR) and median OS was NR (95% CI, 0.57 to NR). One pt with MDS had SD as best response (95% CI, 0.4-64.1). Conclusions: Bomedemstat up to 6.0 mg/kg alone or in combination with ATRA was well-tolerated in pts with high-risk AML or MDS. Change in blast count seen in treated pts could be due to differentiation to monocytes induced by bomedemstat rather than blast count reduction per se. Objective responses were seen in a subset of pts with AML (28%). OS was numerically higher to historical data with standard of care. Bomedemstat is currently under investigation in myeloproliferative neoplasms.

Glucose Metabolism in Acute Myeloid Leukemia Cell Line Is Regulated via Combinational PI3K/AKT/mTOR Pathway Inhibitors.

Metabolism reprogramming is a survival mechanism in acute myeloid leukemia (AML) cells in the tumor microenvironment. Therefore, we investigated the effect of signaling pathway inhibitors on the expression of genes rewired in the metabolic pathway of AML cells. HL-60 cells were treated with Idelalisib, MK-2206, and Everolimus, which respectively are selective inhibitors of phosphatidylinositol-3-kinase (PI3K), AKT, and the mammalian target of rapamycin (mTOR), either individually or in combination. The relative expressions of glucose transporter 1, hexokinase 2, pyruvate kinase, pyruvate dehydrogenase E1, citrate synthase, isocitrate dehydrogenase 2, and hypoxia inducible factor 1 subunit alpha were determined by real-time PCR. The combined treatment of HL-60 cells with Idelalisib, MK-2206, and Everolimus decreased the expression of glucose transporter 1, hexokinase 2, pyruvate kinase M2, pyruvate dehydrogenase E1, citrate synthase, isocitrate dehydrogenase 2, and hypoxia inducible factor 1 subunit alpha. A combination of PI3K/AKT/mTOR pathway inhibitors regulates the expression of genes involved in glycolysis, pyruvate dehydrogenase complex (PDH), and the tricarboxylic acid (TCA) cycle and interferes with metabolic reprogramming and immune evasion mechanisms of AML leukemic cells. Combinational therapy approaches to block these pathways might be a promising and novel therapeutic strategy for targeting the metabolic requirements of AML cells.

Jiyuan oridonin A induces differentiation of acute myeloid leukemia cells including leukemic stem-like cells.

Acute myeloid leukemia (AML) is an aggressive form of hematological neoplasia characterized by failure of myeloid differentiation. AML is a leading cause of death from leukemia. Cytarabine chemotherapy resistance is a major source of refractory/relapsed AML. A major obstacle to the successful treatment of AML results from residual disease maintained by leukemic stem cells (LSCs), which are mostly resistant to conventional chemotherapy. Here, we determined the effect of a natural compound, Jiyuan oridonin A (JOA), on the differentiation blockade in the M2 subtype [particularly t (8;21)] of AML cells, M3 subtype of AML cells (APL cells), and leukemic stem-like cells both in vitro and in vivo. We found that JOA induced cell differentiation and suppressed the colony formation capacity in various AML cell lines (Kasumi-1, KG-1, MUTZ-8, NB4, and HL-60) without eliciting apoptosis. The mechanism of JOA-induced cell differentiation depends on the specificity of cell type. JOA mediated the differentiation of Kasumi-1 cells by activating the hematopoietic cell lineage signaling pathway, while inhibition of c-MYC was involved in the JOA-induced differentiation of NB4 cells. Moreover, JOA was identified to target leukemic stem-like cells by induced cell differentiation in vivo. These findings demonstrated that JOA could inhibit the proliferation of M2 and M3 subtypes of AML cells and leukemic stem-like cells by overcoming the differentiation blockade, which may offer a novel therapeutic strategy for AML to overcome relapse and drug resistance in patients with AML. Our findings highlight the possibility of using compounds like JOA as a promising differentiation-induced agent for the treatment of AML.

The Effects of PI3K/Akt/mTOR Signaling Pathway Inhibitors on the Expression of Immune Checkpoint Ligands in Acute Myeloid Leukemia Cell Line.

Up-regulation of immune checkpoint ligands is considered as one of the most important immune escape mechanisms in acute myeloid leukemia (AML). Herein, we investigate a relationship between the inhibition of PI3K/Akt/mTOR signaling pathways and the regulation of immune checkpoint ligands in AML cells. The HL-60 cell line was treated with idelalisib as PI3K inhibitor, MK-2206 as Akt inhibitor, and everolimus as mTOR inhibitor either in a single or combined format. Cell viability and apoptosis were evaluated using MTT and flow cytometry assays, respectively. The relative expression of PD-L1, galectin-9, and CD155 was determined by real-time PCR. Our findings demonstrated decreased proliferation and increased apoptosis of HL-60 cells after treatment with idelalisib, MK-2206, and everolimus. As expected, the combined treatment showed a more inhibiting effect than the single treatment. Interestingly, our results elucidated that the expression of PD-L1 and Gal-9 but not MK-2206 decreased after treatment with idelalisib and everolimus. Regarding CD155, the expression of this molecule was downregulated after treatment with everolimus, but not idelalisib and MK-2206. However, combined treatment of HL-60 cells with two or three inhibitors decreased the expression levels of PD-L1, Gal-9, and CD155 checkpoint ligands. We showed that PI3K/Akt/mTOR pathway inhibitors not only serve as cytotoxic drugs but also regulate the expression of immune checkpoint ligands and interfere with the immune evasion mechanisms of AML leukemic cells. Combinational treatment approaches to block these pathways might be a promising and novel therapeutic strategy for AML patients via interfering in immune escape mechanisms.

Genetic diversity within leukemia-associated immunophenotype-defined subclones in AML

Acute myeloid leukemia (AML) is a highly heterogeneous disease showing dynamic clonal evolution patterns over time. Various subclones may be present simultaneously and subclones may show a different expansion pattern and respond differently to applied therapies. It is already clear that immunophenotyping and genetic analyses may yield overlapping, but also complementary information. Detailed information on the genetic make-up of immunophenotypically defined subclones is however scarce. We performed error-corrected sequencing for 27 myeloid leukemia driver genes in 86, FACS-sorted immunophenotypically characterized normal and aberrant subfractions in 10 AML patients. We identified three main scenarios. In the first group of patients, the two techniques were equally well characterizing the malignancy. In the second group, most of the isolated populations did not express aberrant immunophenotypes but still harbored several genetic aberrancies, indicating that the information obtained only by immunophenotyping would be incomplete. Vice versa, one patient was identified in which genetic mutations were found only in a small fraction of the immunophenotypically defined malignant populations, indicating that the genetic analysis gave an incomplete picture of the disease. We conclude that currently, characterization of leukemic cells in AML by molecular and immunophenotypic techniques is complementary, and infer that both techniques should be used in parallel in order to obtain the most complete view on the disease.

Treatment with the Recombinant SLIT2 Protein Delays AML Leukemogenesis In Vivo

Accumulating evidence suggest that the axon guidance molecules SLIT and ROBO are not only implicated in physiological process but also in cancer progression. Depending on the type of cancer the SLIT-ROBO axis can either act as a tumor suppressor gene, in which case the SLIT2 promoter site is frequently hypermethylated or as an oncogene, whereby high expression is often associated with poor prognosis. In the context of acute myeloid leukemia (AML), low expression of SLIT2 has been associated with low overall survival (OS) (Golos et al., 2019), while the functional role of SLIT2 remains largely unknown. Recently, we showed that the knockdown of SLIT2 increased cell proliferation of acute promyelocytic leukemia (APL) cells resulting in a more aggressive course of disease progression in vivo using the murine transgenic APL model (Weinhäuser et al., 2020). Here, we aimed to study the functional role of SLIT2 in a more heterogeneous disease, such as AML.Using different publicly available datasets. (GSE58477, normal karyotype blasts: 62, healthy CD34 +: 10; GSE63409, LSC: 14, HSC: 5) we detected increased methylation at the SLIT2 promoter site of AML leukemic cells compared to healthy CD34 + cells suggesting SLIT2 tumor suppressive functions. In addition, we measured decreased levels of SLIT2 in the bone marrow (BM) plasma of AML patients compared to healthy donors. To assess the biological role of SLIT2, we treated AML cell lines (KASUMI1, MV411, and MOLM13) with recombinant SLIT2 (50 ng/mL) in vitro. Administration of SLIT2 reduced AML cell growth, colony formation and induced cell cycle arrest in the G1 phase for all AML cell lines. Conversely, the knockdown of SLIT2 promoted increased THP-1 and OCI-AML3 cell proliferation. Next, we determined whether the treatment with SLIT2 could delay leukemogenesis in vivo using the AML cell line MV4-11. Engraftment was monitored by luciferase bioluminescent signal and NSGS mice were either treated with recombinant SLIT2 using a dose of 25 ng/g of body weight or vehicle (control group). SLIT2 therapy resulted in a lower disease burden, decreased leukemic infiltration in the BM and spleen, reduced spleen size, and increased OS compared to the control group (p<0.05). In conclusion, we showed that SLIT2 methylation is recurrent in AML patients and that the level of SLIT2 in the plasma of AML patients is reduced. Moreover, SLIT2 treatment appears to have a cytostatic effect on different AML cell lines delaying leukemogenesis in vivo. Overall, our study reveals the therapeutic potential of SLIT2 in hematological malignancies, which could be used as an adjuvant in the clinic. DisclosuresNo relevant conflicts of interest to declare.

Protein Arginine Methyltransferase 5 Promotes the Migration of AML Cells by Regulating the Expression of Leukocyte Immunoglobulin-Like Receptor B4.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults with poor prognosis. Especially for AML-M5 type, due to the strong cell migration ability, the possibility of extramedullary invasion is large and widespread, which leads to poor therapeutic effect. Previous studies have found that protein arginine methyltransferase 5 (PRMT5) could promote the proliferation and differentiation of leukemic cells in AML, but its regulation on the invasive ability of AML cells remains unclear. This study was designed to explore the role of PRMT5 in regulating the invasion of AML cells and to investigate the mechanisms. Patient samples were collected for detection of PRMT5 expression level. AML cells were used for exploring the function of PRMT5. The results of clinical samples showed that the expression of PRMT5 was significantly increased in newly diagnosed and recurrent AML patients, and the expression of leukocyte immunoglobulin-like receptor B4 (LILRB4) was positively correlated with the level of PRMT5. In the cell experiment in vitro, we found that when PRMT5 was knocked down, the invasion, migration, and adhesion capacities of MV-4-11 cells and THP-1 cells were decreased, and the mRNA and protein levels of LILRB4 were also decreased. Moreover, we screened related signaling pathways and found that PRMT5 affected the expression of downstream LILRB4 by activating mTOR pathway, which in turn enhanced the invasive ability of AML cells. Taken together, PRMT5 plays an important role in the invasion of AML, which acts via regulating the expression of LILRB4. PRMT5 could act as a potential therapeutic candidate for AML.

Antineoplastic effect of a diphtheria toxin-based nanoparticle targeting acute myeloid leukemia cells overexpressing CXCR4

Nanomedicine has opened an opportunity to improve current clinical practice by enhancing the selectivity in the delivery of antitumor drugs to specific cancer cells. These new strategies are able to bypass toxicity on normal cells increasing the effectiveness of current anticancer treatments. In acute myeloid leukemia (AML) current chemotherapy treatments generate a relevant toxic impact in normal cells and severe side effects or even patient death. In this study, we have designed a self-assembling protein nanoparticle, T22-DITOX-H6, which incorporates a ligand (T22) targeting CXCR4-overexpressing (CXCR4+) cells, and a potent cytotoxic diphtheria toxin domain. CXCR4 is overexpressed in AML leukemic cells and associates with poor prognosis, being, therefore, a relevant clinical target. We demonstrate here that T22-DITOX-H6 induces apoptosis in CXCR4+ leukemic cells through CXCR4-dependent internalization. In addition, repeated T22-DITOX-H6 treatment (10 μg/dose per 10 doses, intravenously injected) in a disseminated AML mouse model (NSG mice intravenously injected with THP-1-Luci cells, n = 10 per group) potently blocks the dissemination of AML cells in bone marrow, spleen and liver of treated mice, without inducing toxicity in healthy tissues. In conclusion, our strategy of selectively ablating CXCR4 positive leukemic cells by administering the T22-DITOX-H6 nanoparticle could be a promising treatment, especially in patients undergoing AML relapse after chemotherapy, in which leukemic cells overexpress CXCR4.

Target-Informed Repurposing of Immunotherapies in AML - a Transcriptome Based Approach for Identifying Immediately Available Therapeutics

Acute Myeloid Leukemia (AML) is an aggressive hematopoietic malignancy with limited therapeutic options and a great need for innovative treatment. However, the identification of novel molecular biomarkers remains challenging, and significant lag time from target discovery to clinical development impedes timely implementation of novel therapies. We previously demonstrated aberrant expression of non-hematopoetic (NH) antigens on leukemic cells in AML; one such antigen, mesothelin (MSLN), has recently begun AML clinical development, providing evidence for the use of NH directed therapeutics in AML. Given this observation, we questioned whether we could build a computational platform to identify misregulated genes in AML that are targeted by currently available immunotherapies in other malignancies. Early phase trial data for these targets will provide vital safety and tolerability information, thereby supporting a rapid path to AML clinical development. To this end, we curated a knowledgebase of antibody-drug conjugate (ADC) and chimeric antigen receptor T-cell (CAR T) therapies by querying all cancer immunotherapy trials registered in the U.S. National Library of Medicine clinical trials database (https://clinicaltrials.gov) and the Journal of Antibody-Drug Conjugates (https://adcreview.com) in November of 2019. In total, our knowledgebase contained 893 clinical trials assessing 141 prospective gene targets. We interrogated transcriptome expression of these targets using ribodepleted RNA-seq data from the TARGET AML and TpAML initiatives (N = 1,394, age &amp;lt;0 to 30 years) and the TCGA LAML cohort (N = 173, age 18 - 88 years). We selected for therapeutic targets expressed in AML by employing a minimum transcript expression threshold of 5 TPM in &amp;gt;50% of our AML cases. This led to the identification of 35 targets of interest for further analysis (Figure 1A). A total of 131 immunotherapies (70 ADCs and 61 CAR Ts) directed against these targets are in various stages of clinical development (Figure 1B). These included 62 AML-directed therapies targeting a total of 13 antigens; among these, CD33, CD123, FLT3, CD117, and CLEC12A have been well described in the context of AML. The remaining 69 therapies (43 ADCs and 26 CAR-Ts) target 30 NH antigens. Nine (30%) of these NH antigens, including CD74, BSG, TFRC, CXCR4, and CD44, all possesed a median of &amp;gt;90 TPM in our AML cohort and were subjects of therapies in Phase I or later. Additionally, these genes were universally expressed across major fusion groups (Figure 1C), suggesting potential as therapeutic targets regardless of age or genetic abnormality. A table of NH targets with high expression in AML is provided in Figure 1D. Conversely, we also identified targets for which expression was uniquely restricted to rare, high-risk, and therapeutically challenging AML variants. Upregulated genes in these variants were identified via differential expression analysis of CBFA2T3-GLIS2, NUP98-NSD1, and NUP98-KDM5A fusions. Genes were filtered using a log fold-change (logFC) threshold of 1 and FDR-adjusted p &amp;lt; 0.05, then cross-referenced with the full knowledgebase to identify 37 rare-variant enriched targets. The top 5 most highly enriched targets (ranked by logFC) were FOLR1, NCAM1, MMP14, TNF, and DLK1. The association of NCAM1 with CBFA2T3-GLIS2 has been previously described. However, FOLR1, a member of the folate receptor family that is overexpressed in epithelial-derived tumors, is a novel target in the context of AML. Like NCAM1, FOLR1 expression in our AML population is almost entirely limited to the CBFA2T3-GLIS2 fusion: 36/43 FOLR1-expressing patients possessed a CBFA2T3-GLIS2 fusion, comprising 94.74% of all CBFA2T3-GLIS2 fusions in our cohort. We screened transcriptome data from a large AML cohort for expression of immunotherapeutic targets with potential utility in AML. This enabled the identification of both broadly expressed and subtype-restricted putative targets. Future expansion of this methodology into other cellular therapies (CAR NK or BiTE) and the examination of new data sources could reveal additional targets with robust expression in AML. Demonstration of cell surface expression of these targets, and appropriate preclinical evidence of efficacy, could lay the groundwork for quickly moving these therapies to clinical trials in AML. Figure Disclosures No relevant conflicts of interest to declare.

PF204 TARP AS IMMUNOTHERAPEUTIC TARGET IN AML EXPRESSED IN THE LSC COMPARTMENT

Background:Acute myeloid leukemia (AML) accounts for 80% of adult and 20% of pediatric leukemias. Despite frequent initial remission, patients exhibit a high relapse risk, probably due to therapy‐resistant leukemic stem cells (LSCs). Particularly the prognosis of FLT3‐ITD harboring patients remains extremely poor.Aims:To identify LSC‐specific targets and develop immunotherapeutic strategies for LSC elimination while assuring salvage of normal hematopoietic stem cells (HSCs).Methods:The GSE 17054 micro‐array dataset was used for transcriptome analysis of CD34+CD38‐ populations sorted from de novo adult AML (LSCs, n = 9) and healthy adults (HSCs, n = 4). We next evaluated transcript expression in CD34+CD38‐ and CD34+CD38+ populations sorted from de novo pediatric AML (pedAML) and healthy controls. An 8x60K human gene expression micro‐array was performed in four pediatric AML (pedAML) (2 FLT3 WT, 2 FLT3‐ITD) and 3 cord blood samples. In addition, qPCR was performed in 13 pedAML, 17 adult AML, and 15 controls (cord blood, normal bone marrow and mobilized blood stem cells). Finally, cell lines of various origin, including 9 AML cell lines, were examined. Protein expression was evaluated by Western blotting and confocal microscopy. Viral transduction was used to generate T‐cell receptor (TCR)‐engineered cytotoxic T‐cells (CTLs), next to HLA‐A∗0201 expressing, TARP overexpression and TARP knockdown AML cell lines. Killing by TCR‐engineered CTLs was evaluated in vitro by cytokine assays, flow cytometry‐ and bioluminescence‐based killing assays.Results:Re‐analysis of the GSE 17054 dataset revealed that the TCRγ chain alternate reading frame protein (TARP) was the most differentially expressed gene between LSC and HSC. Until now, TARP is known as a prostate‐specific marker, also expressed in breast adenocarcinoma. Subsequently, micro‐array and qPCR analysis showed aberrantly high TARP expression within pedAML and adult AML leukemic blasts and LSCs, while absent in normal counterparts. Noteworthy, all FLT3‐ITD harboring pedAML patients (n = 8) showed high TARP expression (P &lt; 0.01), whereas this association was not significant for FLT3‐ITD adult AML (n = 9). No TARP transcripts were identified in B‐ALL, CML nor B‐cell lines, while expression in AML cell lines was significantly higher (P &lt; 0.001). Aberrant TARP expression in cell lines and primary patient samples was confirmed at the protein level and confocal microscopy further corroborated these data. Importantly, cell lines and primary patient samples co‐expressing TARP and HLA‐A∗0201 were efficiently killed by TARP‐TCR engineered CTLs, providing proof‐of‐concept that TARP qualifies as a physiologically relevant target for T‐cell therapy in AML.Summary/Conclusion:We showed that TARP is highly expressed in AML leukemic cells, including LSCs, while absent in normal counterparts. High TARP expression is significantly associated with FLT3‐ITD in pedAML, a marker for poor prognosis. TARP‐TCR engineered CTLs effectively killed TARP+ HLA‐A∗0201+ co‐expressing AML cell lines and primary leukemic cells in vitro. To the best of our knowledge, this is the first report indicating that TARP‐TCR engineered CTLs hold great promise for immunotherapeutic T‐cell therapy in AML.

Mebendazole exhibits potent anti-leukemia activity on acute myeloid leukemia

Acute myeloid leukemia (AML) is one of the most common types of acute leukemia in adults with the lowest survival rate of all leukemia. Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure. Thus, finding new drugs with anti-leukemia activities and minimal side effect is urgently needed. Here through screening more than 1000 drugs approved by the Food and Drug Administration (FDA) of United States, the anthelmintic drug mebendazole (MBZ) was found to inhibit the growth of AML cell lines (THP-1, U937, NB4 and K562) and bone marrow mononuclear cells (BM-MNCs) from AML patients at pharmacologically achievable concentrations. In contrast, similar concentration of MBZ had little inhibitory effect on the growth of normal peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC). In addition, MBZ induced mitotic arrest and mitotic catastrophe in AML cells based on nuclear morphology, cell cycle distribution, mitotic marker analyses and the number of multinucleated cells and apoptotic cells. Furthermore, MBZ treatment inhibited activation of Akt and Erk in AML leukemic cells. Finally, MBZ repressed the progression of leukemic cells in vivo and prolonged survival in AML xenograft mouse model. Taken together, our results suggest that MBZ could be a potential new therapeutic agent for the treatment of AML patients.

Abstract 3337: Targeting acute myeloid leukemia via anti-IL1RAP antibodies

Abstract The current standard of care for Acute Myeloid Leukemia (AML) is largely ineffective in terms of achieving long term remission. This is mostly due to inability to remove all of the leukemic cells, and there is accumulating evidence to support the remaining leukemic cells are Leukemic Stem Cells (LSC) which are resistant to standard chemotherapy. We and others (Barreyro L. et. al. 2012) have discovered that IL-1 receptor accessory protein (IL1RAP) is overexpressed on LSC and their progenitors, but not on normal hematopoietic stem cells (HSC). Moreover, knockdown/knockout of IL1RAP expression greatly decreases growth/survival of AML cells. Thus, we have screened anti-IL1RAP therapeutic antibodies by applying FACS based analysis and Complement-Dependent Cytotoxicity (CDC) assays on both AML cell lines and primary patient samples. In a parallel effort, we also applied a functional IL-1β-induced NF-κB signaling assay to identify function-blocking anti-IL1RAP antibodies. Colony Formation Cell (CFC) assay results demonstrated that lead IL1RAP antibodies can significantly impede growth of both AML blast cells and leukemic stem cell-like cells. In contrast, normal HSCs are unaffected by these antibodies. Lead anti-IL1RAP antibodies also showed tumor growth inhibition in an established EOL-1 orthotopic tumor model. One major challenge of targeting IL1RAP by an antibody is that a large amount of IL1RAP protein is secreted into human serum, serving as a potential “sink” that inhibits IL1RAP antibody efficacy. We addressed this by developing a novel IL1RAP antibody that binds preferentially to the membrane-bound IL1RAP protein as compared to the secreted form. We will discuss different therapeutic development strategies to further advance the utilization of these lead anti-IL1RAP therapeutic antibodies for treating AML and other myeloid leukemic indications. Citation Format: Ping Jiang, Bob Y. Liu, Jen Huang, Jennifer Lu, Sharmili Roy, Madhavi Mishra, Xiaoxian Zhao, Jeffrey Lin, Eric D. Hsi, Jagath R. Junutula. Targeting acute myeloid leukemia via anti-IL1RAP antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3337.

Patient-Tailored Analysis of Minimal Residual Disease in Acute Myeloid Leukemia Using Next Generation Sequencing

Background and aim: The importance of sensitive minimal residual disease (MRD) analysis for determination of response to treatment in acute myeloid leukemia (AML) is becoming increasingly evident. Routinely, this analysis is performed using multiparameter flow cytometry, and in select cases with fusion transcripts using reverse transcription polymerase chain reaction. The drawback with flow cytometry is that it is associated with false negativity due to immunophenotypic shifts during treatment and in pending relapse. In addition, leukemia immunophenotypes often overlap with the normal regenerating bone marrow cell populations. Therefore, other means of identifying remaining leukemic cells are warranted. Leukemic cells in AML are characterized by somatic mutations in recurrently mutated genes as well as in random genes, in most cases as single nucleotide variations (SNVs). We have previously reported that leukemia-specific mutations can be readily identified at the time of diagnosis of AML using exome sequencing of high purity sorted leukemic cells and lymphocytes. The aim here was to show that leukemia-specific mutations identified with exome sequencing at diagnosis can serve as markers for MRD, quantified with targeted deep sequencing, during follow-up.Method: Seventeen cases of AML, age 2-71 years old, were included in the study. Leukemic cells and lymphocytes were sorted using fluorescence activated cell sorting (FACS), from blood or bone marrow at diagnosis of AML. Exome sequencing of sorted cell populations was performed on the Illumina platform. Variant calling was performed with Mutect for SNVs and with Strelka and Varscan for short insertions/deletions. The data was subjected to an in-house statistical algorithm to identify variants present in all leukemic cells and thus suitable for MRD analysis. For targeted deep sequencing, the Truseq-library system was used for in-house PCR and sequencing on the Illumina Miseq platform (2x150 bp). The acquired reads were stitched using PEAR, aligned to the human reference genome and the resulting alignments were analyzed with in-house scripts with respect to specific SNVs and NPM1 insertion.Results: Exome sequencing of the paired leukemia/lymphocyte samples identified 240 leukemia-specific SNVs (14 (0-29) per case (median, range) and 22 small insertions and deletions (1 (0-5) per case). The most common type of mutation was, as expected, substitution of cytosine to thymine (CàT). The number of leukemia specific SNVs correlated with age (r=0.76, p<0.001). Mutations suitable for MRD analysis were identified in all but one of the investigated AML cases. Targeted deep sequencing of leukemic cells in serial dilutions established linearity down to a determined variant allele frequency (VAF) of 0.025% for SNVs and of 0.016% for insertion in NPM1. The level of detection (mean+3SD of normal samples) was VAF 0.025% for SNVs and VAF 0.007% for insertion in NPM1. Targeted deep sequencing was then performed on DNA prepared from follow-up bone marrow slides from a patient with AML with mutations suitable for MRD analysis according to our algorithm. Targeted deep sequencing of three SNVs (in the genes CPS1, ITGB7 and FAM193A) and NPM1 type A mutation could detect mutations at all eight time points tested. There were strong correlations between the detected mutation load of the SNVs and the NPM1 type A mutation and all four mutations were present at relapse 10 months after diagnosis. Targeted deep sequencing of SNVs was in this case more sensitive and robust than multiparameter flow cytometry, which could not detect leukemic cells (<0.1% of all cells) at two of the tested time points (5 and 8 months after diagnosis) and showed a completely switched immunophenotype of leukemic cells at relapse.Conclusions: Exome sequencing of high purity sorted leukemic cells and lymphocytes at the time of diagnosis of AML can identify leukemia-specific mutations suitable for MRD analysis. With targeted deep sequencing of leukemia-specific SNVs identified in this manner, leukemic cell burden can be estimated with high sensitivity during follow-up. The method could be used for patient-tailored MRD analysis in AML. DisclosuresNo relevant conflicts of interest to declare.

Favorable clinical outcome and unique characteristics in association with Twist1 overexpression in de novo acute myeloid leukemia.

Epithelial–mesenchymal transition (EMT) is a critical process for inducing stem-like properties of epithelial cancer cells. However, the role of EMT inducers in hematological malignancies is unknown. Twist1, an EMT inducer necessary for cell migration, has recently been found to have transcriptionally regulatory activity on the expression of Bmi1, and these two are capable of promoting tumorigenesis in a synergized manner. Knowing that Bmi1 expression is essential for maintenance of leukemic stem cells, we speculate that Twist1 might govern the pathogenesis of acute myeloid leukemia (AML) development as well. We found that upregulated Twist1 increased Bmi1 expression in AML and endued leukemic cells a higher proliferative potential and increased resistance to apoptosis. In primary AML samples, there was strong positive correlation between the expression levels of Twist1 and Bmi1. AML patients whose leukemic blasts harbored overexpressed Twist1 had a more aggressive clinical phenotype, but they were more likely to have a better clinical outcome after standard therapy. In vitro studies confirmed that Twist1-overexpressing leukemic cells were more susceptible to cytarabine, but not daunorubicin, cytotoxicity. Our findings suggest that, in a subset of AML patients, Twist1 has a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes.

A SALL4/MLL/HOXA9 pathway in murine and human myeloid leukemogenesis

The embryonic self-renewal factor SALL4 has been implicated in the development of human acute myeloid leukemia (AML). Transgenic mice expressing the human SALL4B allele develop AML, which indicates that this molecule contributes to leukemia development and maintenance. However, the underlying mechanism of SALL4-dependent AML progression is unknown. Using SALL4B transgenic mice, we observed that HoxA9 was significantly upregulated in SALL4B leukemic cells compared with wild-type controls. Downregulation of HoxA9 in SALL4B leukemic cells led to decreased replating capacity in vitro and delayed AML development in recipient mice. In primary human AML cells, downregulation of SALL4 led to decreased HOXA9 expression and enhanced apoptosis. We found that SALL4 bound a specific region of the HOXA9 promoter in leukemic cells. SALL4 overexpression led to enhanced binding of histone activation markers at the HOXA9 promoter region, as well as increased HOXA9 expression in these cells. Furthermore, we observed that SALL4 interacted with mixed-lineage leukemia (MLL) and co-occupied the HOXA9 promoter region with MLL in AML leukemic cells, which suggests that a SALL4/MLL pathway may control HOXA9 expression. In summary, our findings revealed a molecular mechanism for SALL4 function in leukemogenesis and suggest that targeting of the SALL4/MLL/HOXA9 pathway would be an innovative approach in treating AML.

SAR103168: a tyrosine kinase inhibitor with therapeutic potential in myeloid leukemias

SAR103168, a tyrosine kinase inhibitor of the pyrido [2,3-d] pyridimidine subclass, inhibited the kinase activities of the entire Src kinase family, Abl kinase, angiogenic receptor kinases (vascular endothelial growth factor receptor [VEGFR] 1 and 2), Tie2, platelet derived growth factor (PDGF), fibroblast growth factor receptor (FGFR) 1 and 3, and epidermal growth factor receptor (EGFR). SAR103168 was a potent Src inhibitor, with 50% inhibitory concentration (IC50) = 0.65 ± 0.02 nM (at 100 μM ATP), targeting the auto-phosphorylation of the kinase domain (Src260-535) and activity of the phosphorylated kinase. Phosphorylation of Src, Lyn and Src downstream signaling pathways (PYK2, P-130CAS, FAK, JNK and MAPK) were inhibited in a dose-dependent manner. SAR103168 inhibited the phosphorylation of STAT5 in KG1 cells and fresh cells from patients with acute myeloid leukemia (AML). SAR103168 inhibited proliferation and induced apoptosis in acute and chronic myeloid leukemic cells at nanomolar IC50. SAR103168 induced anti-proliferation of leukemic progenitors (CFU-L) from 29 patients with AML, and > 85% of AML patient samples were sensitive to SAR103168. These antagonist activities of SAR103168 were independent of FLT3 expression. SAR103168 treatment was effective in 50% of high-risk patient samples carrying chromosome 7 abnormalities or complex rearrangement. SAR103168 administration (intravenous or oral) impaired tumor growth and induced tumor regression in animals bearing human AML leukemic cells, correlating with potent inhibition of Src downstream signaling pathways in AML tumors. SAR103168 showed potent anti-tumor activity in SCID (severe combined immunodeficiency) mice bearing AML (KG1, EOL-1, Kasumi-1, CTV1) and chronic myeloid leukemia (CML) (K562) tumors. The combination of cytarabine and SAR103168 showed synergistic activity in AML and CML tumor models. These results highlight the therapeutic potential of SAR103168 in myeloid leukemias and support the rationale for clinical investigations.

Reactivating PP2A by FTY720 as a Novel therapy for AML with C‐KIT tyrosine kinase domain mutation

The tyrosine kinase domain (TKD) mutations of receptor tyrosine kinase C-KIT are associated with a poor prognosis in acute myeloid leukemia (AML). However, the underlying mechanisms are not fully understood. We found the activity of protein phosphatase 2A (PP2A), a human tumor suppressor whose dysfunction contributes to malignant cell behavior, was significantly decreased in AML subgroups harboring C-KIT/D816V and AML cell line Kasumi-1 bearing C-KIT/N822K mutation. Primary AML cells and various AML cell lines were treated with PP2A activator FTY720. FTY720 showed a toxic effect in all leukemic cells, especially for cells harboring C-KIT/TKD mutation. Furthermore, FTY720-induced toxicity in AML leukemic cells was mediated by restoration of PP2A activity, via down-regulation of PP2A inhibitor SET, dephosporylation of PP2A-C(TYR307), and up-regulation of relevant PP2A subunit A and B55α. Our research indicates that the decreased PP2A activity in AML harboring C-KIT/TKD mutation may make the restoration of PP2A activity a novel therapy for AML patients with C-KIT/TKD mutation.

Phenomenon of evolution of clonal chromosomal abnormalities in childhood acute myeloid leukemia

Analysis of chromosomal abnormalities in bone marrow cells in 116 children with diagnosis of acute myeloid leukemia (AML) was performed. Frequency of evolution of clonal chromosome abnormalities in AML constituted 42,3%. The most abundant among them were numerical abnormalities of chromosomes 8, 9, and 21 as well as secondary structural abnormalities in region 12p12, 9p22, 9q22, 9q34, 11q14-23, and 16q22. Numerical abnormalities were registered in 26,7% cases. The basic mechanism of leukemic clone evolution was trisomy, deletion and monosomy. The frequency of evolution was 7 times higher in the age group up to 2 years and twice higher in the age group up to 5 years. The high frequency of evolution was established at t(15;17)(q22;q22) and the absence at inv(16)(p13q22). The patients with clonal evolution died earlier, before reaching remission, that can be connected with heavy initial state and high frequency of relapse. Conception of abnormality clone evolution was proposed at some stages: I--appearance of balanced rearrangement; II--trisomy; III--lose of chromosomal material. Appearance of unbalanced genome in evolution possess an advantage in proliferate activity and can be connected with the answer on chemotherapy. Identity of abnormal chromosome structure at diagnosis and relapse of disease can be an evidence of the influence of chemical agent on establishment of some types of evolution of chromosome abnormalities in leukemic cells in AML in children.

Influence of high expression of Smac/DIABLO protein on the clinical outcome in acute myeloid leukemia patients

The role of the Smac/DIABLO protein, a novel apoptosis agonist, in acute myeloid leukemia (AML) is not clearly determined. The expression of Smac/DIABLO protein in AML leukemic cells and its relationship with clinical outcome was evaluated in this study. The intracellular expression of Smac/DIABLO protein was assessed using multi-color flow cytometry in 71 newly diagnosed AML patients treated with conventional chemotherapy. It was found that the high expression of Smac/DIABLO protein was an independent prognostic factor in terms of higher complete remission rate ( p < 0.001) and longer overall survival ( p = 0.003). Moreover the low expression of Smac/DIABLO protein was associated with poor karyotype.