Adult Acute Myeloid Leukemia Cells Research Articles

Inhibition of NRF2 enhances the acute myeloid leukemia cell death induced by venetoclax via the ferroptosis pathway

Venetoclax, an inhibitor that selectively targets B cell lymphoma-2 (BCL-2) that has been approved for treating adult acute myeloid leukemia (AML) in combination with hypomethylating agents. However, its short duration of response and emergence of resistance are significant issues. In this study, we found that the sensitivity of AML cells to venetoclax was considerably enhanced by ML385, an inhibitor of the ferroptosis factor nuclear transcription factor erythroid 2-related factor 2 (NRF2). Using AML samples, we verified that NRF2 and its target gene ferritin heavy chain 1 (FTH1) were highly expressed in patients with AML and correlated with poor prognosis. Downregulation of NRF2 could inhibit FTH1 expression and significantly enhance the venetoclax-induced labile iron pool and lipid peroxidation. By contrast, NRF2 overexpression or administration of the reactive oxygen species inhibitor N-acetylcysteine and vitamin E could effectively suppress the anti-AML effects of ML385+venetoclax. Furthermore, the ferroptosis inducer erastin increased the anti-AML effects of venetoclax. Our study demonstrated that NRF2 inhibition could enhance the AML cell death induced by venetoclax via the ferroptosis pathway. Thus, the combination of ML385 with venetoclax may offer a favorable strategy for AML treatment.

DELE1 haploinsufficiency causes resistance to mitochondrial stress-induced apoptosis in monosomy 5/del(5q) AML

Monosomy 5 and deletions of the chromosome 5q (−5/del(5q)) are recurrent events in de novo adult acute myeloid leukemia (AML), reaching up to 40% of cases in secondary AML. These chromosome anomalies are associated with TP53 mutations and with very poor prognosis. Using the large Leucegene genomic and transcriptomic dataset composed of 48 −5/del(5q) patient specimens and 367 control AML, we identified DELE1 – located in the common deleted region – as the most consistently downregulated gene in these leukemias. DELE1 encodes a mitochondrial protein recently characterized as the relay of mitochondrial stress to the cytosol through a newly defined OMA1-DELE1-HRI pathway which ultimately leads to the activation of ATF4, the master transcription factor of the integrated stress response. Here, we showed that the partial loss of DELE1 expression observed in −5/del(5q) patients was sufficient to significantly reduce the sensitivity to mitochondrial stress in AML cells. Overall, our results suggest that DELE1 haploinsufficiency could represent a new driver mechanism in −5/del(5q) AML.

Myeloid Master Regulators PU.1 and CEBPA Suppress Leukemogenesis through Downregulating Cell Cycle Related Genes in NPM1 Mutated Leukemia

Nucleophosmin1 mutation (NPM1c) is the most common mutation in adult acute myeloid leukemia (AML) and known as a driver mutation causing leukemogenesis through upregulating homeotic gene transcription. Mutated NPM1 is reported to mis-localize in the cytoplasm. However, how cytoplasmic NPM1c regulates leukemic transcription remains poorly understood. We previously reported that NPM1c reprograms homeotic and leukemogenic transcription through reshaping CTCF-defined three-dimensional topology associated domain (TAD). Also, re-localization of cytoplasmic NPM1c to the nucleus switches MIZ1/MYC repressive transcription regulatory axis to MIZ1/NPM1 active transcription regulatory axis at CDKN locus and CEBPA locus leading to cell cycle arrest and myeloid differentiation in NPM1 mutated AML. In this study, we are focused on regulation of myeloid differentiation and hypothesized upregulation of myeloid master regulators, PU.1 and CEBPA may suppress the leukemogenic transcription and induce myeloid differentiation in NPM1 mutated AML cells. To confirm NPM1c regulates TAD formation at CEBPA locus, we performed HiC-seq in OCI-AML3 with nuclear re-localized NPM1c using XPO1 inhibitor treatment and NPM1-wildtype overexpression. Nuclear re-localized NPM1 upregulates CTCF-defined TAD interaction at CEBPA locus and upregulates CEBPA and CEBPD transcription which indicates NPM1c regulates CEBPA transcription via altering CEBPA TAD formation. We then test whether activation of CEBPA or SPI1 in NPM1 C+ AML cells induced myeloid differentiation and blocked NPM1 C+ driven leukemogenesis. We transcriptionally activated SPI1 or CEBPA gene in OCI-AML3 using CRISPR-dCas9-VP160 system to determine how these myeloid regulators changed the leukemic transcription and leukemic cell phenotype. PU.1- or CEBPA- activated OCI-AML3 reduces cell proliferation, especially affecting the G2/M phase in cell cycle. They have monocyte-like nuclei with large cytoplasm and induce myeloid differentiation with upregulating both of CD11b and CD14 myeloid cell surface markers. Moreover, compared to parental OCI-AML3 cells, Pu.1- or CEBPA-activated cells showed lower colony formation ability and in xenograft mouse models produced diminished leukemic symptoms and prolonged survival. We further conducted RNA-seq to explore how PU.1 or CEBPA changed the gene transcription leading to the blockage of leukemogenesis. Between PU.1- and CEBPA-activated cells, 58% of differentially expressed genes overlap and GO or GSEA analysis revealed that both of them downregulates G2M checkpoint/mitotic-spindle cell cycle related gene sets, indicating that PU.1 or CEBPA activation controls cell cycle control rather than myeloid cell differentiation in NPM1 C+ AML. In summary, our data revealed that re-localization of NPM1 C+ into nucleus activates master myeloid transcription factors, PU.1 and CEBPA, which, in turn, suppress leukemogenesis via inducing cell cycle arrest and myeloid cell differentiation in NPM1 C+ AML.

Oncometabolite-Independent Anti-Apoptotic Feature Via Phospholipid Metabolic Adaptation in IDH2 Mutant AML Cells

Isocitrate dehydrogenase (IDH) gene mutation is found in approximately 20% of adult acute myeloid leukemia (AML) patients. IDH gene mutations give a pathological enzymatic activity producing oncometabolite, which impair hematopoietic differentiation while promoting leukemogenesis via epigenetic changes. Targeting mutant (mut) form of IDH enzymes, small molecule inhibitors have been developed. Among these inhibitors, AG-221 (enasidenib), which is the first-in-class, selective and orally available mut IDH2 inhibitor (Yen et al., Cancer Discov. 2017), has been tested in clinical trial. The clinical trial showed overall response rate was approximately 40% and more than half of the patient didn't respond to the treatment. This clinical study also showed clear correlation between clearance of IDH2 mut clones and complete clinical response rate (Stein et al., Blood 2019). Relapse cases under the AG-221 treatment, in which additional mutation in IDH2 or IDH1 gene occurred in the residual IDH2 mut clone have been reported (Intlekofer et al., Nature 2018, Harding et al., Cancer Discov. 2018). These reports suggested that IDH2 mut cells have an oncometabolite-independent survival advantage which contributes to refractoriness and relapse. However, most of the studies on IDH mut AML have been focused on oncometabolite-related mechanisms and oncometabolite-independent mechanism has not been investigated in detail so far. IDH gene mutations frequently coexist with other oncogenic mutations in AML patient cells, which make it difficult to analyze the precise role of IDH gene mutation in leukemogenesis. To address this issue, we utilized the cytokine-dependent AML cell line, TF-1 cells and its isogenic cell line with IDH2 gene mutation, which showed cytokine independent growth. Though AG-221 suppressed the cytokine independent growth in IDH2 mut cells, the growth of AG-221 treated IDH2 mut cells remained significantly faster than IDH2 wild type (WT) cells. These data indicated that IDH2 mut cells have a growth advantage machinery in an oncometabolite-independent manner. Metabolome analysis clearly separated AG-221 treated IDH2 mut cells from IDH2 WT cells and identified that inositol phosphate metabolism pathway showed significant difference between these two groups. High throughput drug screening using metabolic inhibitors also revealed that inositol phosphate related enzyme, phospholipase C (PLC) specific inhibitor promoted IDH2 mut cell proliferation. These data suggested that suppressed PLC activity induced oncometabolite-independent growth advantage in IDH2 mut AML cells. Since PLC indirectly release free arachidonic acid (AA) from phospholipid bilayer of cellular membrane and intracellular AA level has positive correlation to apoptosis (Cao et al. PNAS 2000), we analyzed intracellular AA and apoptosis in IDH2 mut cells. As expected, these analyses showed significantly lower intracellular AA and reduced Annexin V+ apoptotic cells in IDH2 mut cells irrespective of AG-221 treatment. Supporting these data, PLC activity related gene sets were negatively enriched and anti-apoptosis related genes were positively enriched in IDH2 mut cells in RNA seq analysis. Based on these novel mechanisms, we tested FDA-approved anti-inflammatory drugs, COX-2 and 5-LOX specific inhibitors (celecoxib and zileuton) to upregulate intracellular AA and induce apoptosis of TF-1 cells in vitro and in vivo xenograft. These drugs in combination with AG-221 successfully suppressed IDH2 mut cell proliferation and induced apoptosis both in vitro and in vivo. The mice treated with these triple drugs showed no sign of cytopenia nor therapy-associated complication, securing safety of this combination therapy. In this study, we identified unappreciated oncometabolite-independent anti-apoptotic feature acquisition via phospholipid metabolic adaptation in IDH2 mut AML cells. Moreover, targeting this metabolic adaptation, we identified that FDA-approved anti-inflammatory drugs in combination with mut IDH2 inhibitor could eradicate IDH2 mut AML cells by efficiently inducing apoptosis. Since these anti-inflammatory drugs have a track record of safety in clinical practice, the triple drug combination therapy could be an alternative therapeutic option for the IDH2 mut AML patients who couldn't tolerate existing therapy.

RNAi-Mediated Screen of Primary AML Cells Nominates MDM4 as a Therapeutic Target in NK-AML with DNMT3A Mutations.

DNA-methyltransferase 3A (DNMT3A) mutations belong to the most frequent genetic aberrations found in adult acute myeloid leukemia (AML). Recent evidence suggests that these mutations arise early in leukemogenesis, marking leukemic progenitors and stem cells, and persist through consolidation chemotherapy, providing a pool for AML relapse. Currently, there are no therapeutic approaches directed specifically against this cell population. To unravel therapeutically actionable targets in mutant DNMT3A-driven AML cells, we have performed a focused RNAi screen in a panel of 30 primary AML samples, all carrying a DNMT3A R882 mutation. As one of the strongest hits, we identified MDM4 as a gene essential for proliferation of primary DNMT3AWT/R882X AML cells. We analyzed a publicly available RNA-Seq dataset of primary normal karyotype (NK) AML samples and found a trend towards MDM4 transcript overexpression particularly in DNMT3A-mutant samples. Moreover, we found that the MDM2/4 inhibitor ALRN-6924 impairs growth of DNMT3AWT/R882X primary cells in vitro by inducing cell cycle arrest through upregulation of p53 target genes. Our results suggest that MDM4 inhibition is a potential target in NK-AML patients bearing DNMT3A R882X mutations.

Prognostic Impacts of Additional Cytogenetic Abnormalities Acquired at the First Relapse in Adult Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplant in Second Complete Remission

Background: Additional cytogenetic abnormalities (ACA), the most frequent form of cytogenetic changes, are considered as a result of genetic instability and clonal evolution of leukemia cells. Recently, we described that ACA at the first relapse was associated with the significantly lower second complete remission (CR2) rate and poor survival in adult acute myeloid leukemia (AML) patients (Hematol Oncol. 2018;36:252-257). However, the prognostic impact of ACA after allogeneic stem cell transplant (allo-SCT) has not been elucidated in adult AML patients. Patients and methods: Of the 145 adult AML patients who underwent the first allo-SCT in CR2 between 1997 and 2019, 98 patients whose cytogenetic abnormality data both at diagnosis and at the first relapse were available were included in this study. Cytogenetic changes between at diagnosis and the first relapse were classified into four groups: (1) no change, (2) ACA was acquired at the first relapse, (3) cytogenetic abnormalities observed at diagnosis were reduced or had disappeared at the first relapse, and (4) cytogenetic abnormalities observed at diagnosis were reduced or had disappeared, and completely different ACA was acquired at the first relapse. In this study, groups 2 and 4 were defined as ACA acquisition. Overall survival (OS) was defined as the interval from the date of transplant to the date of death. Fisher's exact test was used to compare binary variables. The cumulative incidence (CI) of non-relapse mortality (NRM) and relapse were evaluated with Gray's test, considering relapse and NRM as a competing risk, respectively. OS was estimated with the Kaplan-Meier method and compared using the log-rank test. Factors associated with at least borderline significance (p < 0.20) in univariate analyses were subjected to multivariate analysis. The Cox and Fine-Gray proportional hazard model were used for multivariate analysis of prognostic and risk factors, respectively. Values of p < 0.05 were considered to indicate statistical significance. Results: Of the 98 patients included in this study, 57 were male, and 41 were female. The median age at transplant was 45 years (range, 17-71 years). The median duration of CR1 was 12.4 months (range, 1.3-70.3 months) and cytogenetic risk groups were good, intermediate, and poor in 26 (27%), 70 (71%), and two patients (2%), respectively. Donor types were related, unrelated, and cord blood in 23 (24%), 59 (60%), and 16 patients (16%), respectively, and 86 (87%) and 61 patients (62%) were conditioned with myeloablative and total body irradiation-containing regimens, respectively. According to the definition described above, 20 patients (20%) acquired ACA at the first relapse. There was no significant difference in baseline characteristics and transplant procedures between patients with and without ACA acquisition. The OS rates were not significantly different between two groups (55% vs. 72% at three years after transplant; p = 0.28). The CI of relapse was significantly higher in patients with ACA acquisition than those without ACA acquisition (59% vs. 15%; p < 0.01), while the CI of NRM were not significantly different between two groups (5% vs. 19%; p = 0.17). Multivariate analysis for OS revealed that age over 50 years (hazard ratio [HR] = 2.4; p < 0.01), but not ACA acquisition, was identified as an independent prognostic factor. ACA acquisition (HR = 4.7; p < 0.01) was extracted as an independent risk factor of relapse, while use of reduced intensity conditioning regimens (HR = 3.1; p = 0.03) and more than or equal to 1 of performance status at transplant (HR = 2.7; p = 0.04) showed independent risks of NRM. The similar OS rates between two groups might resulted from an offset of the lower relapse rate with the higher NRM rate in patients without ACA acquisition despite not reaching statistical significance. This increasing NRM rates in those without ACA acquisition was potentially associated with use of reduced intensity conditioning regimens in larger proportion (0% vs. 15%; p = 0.12). Conclusion: These findings suggested that ACA acquisition at the first relapse was associated with a higher risk of relapse even after allo-SCT in CR2 in adult AML patients. As AML cells with ACA acquisition was resistant to not only chemotherapy but also graft-versus-leukemia effect, innovative therapeutic strategy is warranted. DisclosuresHanda: Janssen: Honoraria; Kyowa Kirin: Research Funding; Takeda: Honoraria, Research Funding; BMS: Honoraria; Chugai: Research Funding; Ono: Honoraria; Sanofi: Honoraria, Research Funding; Abbvie: Honoraria; MSD: Research Funding; Shionogi: Research Funding; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Research Funding.

RNAi prodrugs decrease elevated mRNA levels of Polo-like kinase 1 in ex vivo cultured primary cells from pediatric acute myeloid leukemia patients.

Polo-like kinase 1 (Plk1) is an important regulator of the cell cycle and it is frequently overexpressed in cancer cells. Several small molecule inhibitors have been developed to target Plk1 and some of them have reached clinical trials in adults with acute myeloid leukemia (AML). Pediatric AML patients have a poor prognosis and survivors suffer from long-term side effects. As adult AML cells have an elevated expression of Plk1, AML is a disease candidate for Plk1 inhibition. However, the relative success of clinical trials have been hampered by adverse reactions. Herein, PLK1-targeting RNA interference (RNAi) prodrugs that enter cells without a transfection reagent are used to target PLK1 selectively in primary cells from pediatric AML patients. We show that PLK1 and PLK4 mRNA expression are significantly higher in pediatric AML patients when compared to healthy donors and that PLK1 is downregulated by on average 50% using RNAi prodrugs without a significant effect on other PLK family members. In addition, the RNAi prodrug-induced decrease in PLK1 can be used to potentiate the effect of cytarabine. In summary, PLK1-targeting RNAi prodrugs can decrease the elevated levels of PLK1 in primary cells from pediatric AML patients and sensitize pediatric AML cells to chemotherapeutics.

TBC1D16 predicts chemosensitivity and prognosis in adult acute myeloid leukemia (AML) patients

Acute myeloid leukemia (AML) is a hematopoietic disease with poor survival. Chemotherapy resistance is one of the determinant factors influencing AML prognosis. To identify genes possibly affecting the drug responses in AML, the Illumina Infinium MethylationEPIC (850K) was used to screen for differential DNA methylation loci between patients achieved complete remission (CR) or not (non-CR) after induction therapy in 37 AML patients. Then, 32 differentially methylated sites (DMS) were selected for replication in another 86 AML patients by next-generation sequencing. Nine sites including cg03988660, cg16804603, cg18166936, cg11308319, cg09095403, cg18493214, cg01443536, cg16030878 and cg10143426 were replicated. Analysis of the Gene Expression Omnibus (GEO) database showed that mRNA expression of TBC1D16 and HDAC4 was associated with AML prognosis. Methylation level of the cg16030878 in TBC1D16 3′-UTR correlated positively with TBC1D16 mRNA expression in samples both in the TCGA database and clinically collected in the study. Both higher cg16030878 methylation and higher TBC1D16 mRNA expression were associated with increased risk of non-CR and worse overall survival (OS) in AML patients. In AML cells, knockdown of TBC1D16 decreased cell proliferation and ERK phosphorylation levels, as well as increased sensitivity to mitoxantrone and decitabine indicated by IC50. In patients with combined use of decitabine, those patients with CR showed significantly lower TBC1D16 mRNA expression. On the contrary, knockdown of TBC1D16 resulted in decreased sensitivity to cytarabine in U937 cells. Our findings implicated that TBC1D16 is a potential predictor for chemosensitivity and prognosis in adult AML patients.

A narrative review of central nervous system involvement in acute leukemias.

Acute leukemias (both myeloid and lymphoblastic) are a group of diseases for which each year more successful therapies are implemented. However, in a subset of cases the overall survival (OS) is still exceptionally low due to the infiltration of leukemic cells in the central nervous system (CNS) and the subsequent formation of brain tumors. The CNS involvement is more common in acute lymphocytic leukemia (ALL), than in adult acute myeloid leukemia (AML), although the rates for the second case might be underestimated. The main reasons for CNS invasion are related to the expression of specific adhesion molecules (VLA-4, ICAM-1, VCAM, L-selectin, PECAM-1, CD18, LFA-1, CD58, CD44, CXCL12) by a subpopulation of leukemic cells, called “sticky cells” which have the ability to interact and adhere to endothelial cells. Moreover, the microenvironment becomes hypoxic and together with secretion of VEGF-A by ALL or AML cells the permeability of vasculature in the bone marrow increases, coupled with the disruption of blood brain barrier. There is a single subpopulation of leukemia cells, called leukemia stem cells (LSCs) that is able to resist in the new microenvironment due to its high adaptability. The LCSs enter into the arachnoid, migrate, and intensively proliferate in cerebrospinal fluid (CSF) and consequently infiltrate perivascular spaces and brain parenchyma. Moreover, the CNS is an immune privileged site that also protects leukemic cells from chemotherapy. CD56/NCAM is the most important surface molecule often overexpressed by leukemic stem cells that offers them the ability to infiltrate in the CNS. Although asymptomatic or with unspecific symptoms, CNS leukemia should be assessed in both AML/ALL patients, through a combination of flow cytometry and cytological analysis of CSF. Intrathecal therapy (ITT) is a preventive measure for CNS involvement in AML and ALL, still much research is needed in finding the appropriate target that would dramatically lower CNS involvement in acute leukemia.

Inducible MyD88 Signaling Enhances the Anti-AML Activity of BiTE-Secreting ENG T-Cells

Background: Pediatric and adult acute myeloid Leukemia (AML) is a disease with poor prognosis due to its high relapse rate and treatment related mortality. Adoptive immunotherapy has the potential to improve outcomes and CD123 present a promising immunotherapy target for AML. CD123-specific engager (ENG) T-cells are cells genetically modified to secrete bispecific T-cell engagers (BiTEs) that recognize CD3+ on T-cells and CD123+ on AML. We had previously shown that inclusion of an inducible MyD88/CD40 costimulatory molecule, which is activated by a chemical inducer of dimerization (CID), improved anti-tumor effect against CD123+ targets in vitro. We now wanted to determine, which component of the costimulatory molecule is critical for the improved effector function of CD123-ENG T cells and perform in vivo studies. Methods: We generated a panel of retroviral vectors encoding CD20 as a safety switch, CD123-ENG, and an inducible costimulatory molecule consisting of a myristoylation sequence, FKBP dimerizer domains and one of three signaling costimulatory domains (MyD88 (iM) ,CD40(iC) or MyD88/CD40(iMC)). A vector specific for CD19 (CD20.CD19-ENG.iMC) and non-transduced (NT) T-cells served as controls. We genetically modified T-cells using a retroviral transduction protocol and evaluated their effector function +/- CID. We assessed the effector function of transduced T-cells using flow cytometry, ELISA and luciferase based cytotoxicity assays. We evaluated antitumor activity and persistence in vivo in a xenograft AML model. Results: We successfully generated CD20.CD123-ENG.iM, CD20.CD123-ENG.iC and CD20.CD123-ENG.iMC T-cells. Transduction efficiency ranged from 45 to 70% (±5). The immunophenotype of CD123-ENG T-cells was predominandly CD8+ effector memory. CD20.CD123-ENG T-cells expressing inducible costimulatory molecules secreted higher levels of CD123-specific BiTEs, IL2 and IFNγ in comparison to CD20.CD123-ENG T-cells when cocultured with of MOLM-13 (CD123+) and CID (p&lt;0.005, N=3). In the presence of CID, CD20.CD123-ENG.iM or CD20.CD123-ENG.iMC T-cells were able to sequentially kill AML cells for up to 5 stimulations, as opposed to CD20.CD123 ENG, CD20.CD123 ENG.iC and NT T-cells (n=3, E:T=1:1). We evaluated the in vivo anti-AML activity of ENG T cells using an AML cell line expressing firefly luciferase (MOLM-13.ffluc) to allow for serial bioluminescence imaging. MOLM-13.ffluc bearing mice were treated with one i.v. dose of effector cells (CD20.CD123-ENG, CD20.CD123-ENG iM, CD20.CD123-ENG.iC, CD20.CD123-ENG.iMC or NT T-cells ) and 2 doses/week of CID for 2 weeks (100mg/mice, i.p.). CD20.CD123-ENG.iMC and iM T-cells had potent anti-leukemic activity, resulting in a significant survival advantage in comparison to CD20.CD123-ENG T-cells group (P&lt;0.005, n=10 animals per group). Conclusion: We demonstrate here that inducible MyD88/CD40 costimulation significantly enhances the anti-AML activity of CD123-ENG T cells in vitro and in xenograft models. Mechanistic studies revealed that MyD88 costimulation is critical for the observed benefit. Thus, genetically modifying T-cells to express BiTEs and an inducible MyD88 costimulatory molecule may present a promising cell product for the immunotherapy of CD123+ hematological malignancies. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents &amp; Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:TESSA Therapeutics: Other: Research Collaboration; Tidal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; EMD Serono: Honoraria; ViraCyte: Consultancy; NHLBI: Research Funding; ASSISI fundation of Memphis: Research Funding; California Institute for Regenerative Medicine: Research Funding; Patents and patent applications in the fields of T-cell &amp; Gene therapy for cancer: Patents &amp; Royalties; America Lebanese Syrian Associated Charities: Research Funding. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents &amp; Royalties: Patent Applications in the Fields of Cell and Gene Therapy .

Flow cytometric analysis of CD64 expression pattern and density in the diagnosis of acute promyelocytic leukemia: a multi-center study in Shanghai, China.

No unified immunophenotypic profiles and corresponding analytic strategies have been established for the rapid diagnosis of acute promyelocytic leukemia (APL) using flow cytometry (FCM). Here we describe a characteristic immunophenotypic panel that can rapidly and accurately distinguish APL from other types of adult acute myeloid leukemia (AML) using only FCM. By comparing APL cells and non-APL AML cells that share APL common immunophenotypes (CD34−CD117+HLA−DR−) we found that CD64 was a significant factor that differentiated APL from other AMLs. Further retrospective analyses of 205 APL and 629 non-APL AML patients from different hematology centers showed that either the CD64dim and homoCD13+homo CD33+homoMPO+ (myeloperoxidase) CD11c− panel or the CD64dim and homoCD13+homo CD33+homoMPO+ CD11c+CD10−CD117+ SSChigh (high side scatter signal) panel could distinguish APL from non-APL AML patients with nearly 100% sensitivity, specificity and accuracy. Moreover, relative quantification of CD64 expression enhanced the applicability of our APL diagnostic immunophenotypic panels (ADI-panels) in different hematology centers. Application of the ADI-panels will decrease diagnosis time and improve personalized treatment for APL, a life-threatening disease with very rapid progression.

Abstract PR09: APOBEC3A sensitizes leukemia cells to inhibitors of the replication checkpoint

Abstract The human APOBEC3 family of DNA-cytosine deaminases comprises seven members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 enzymes are best defined by their capacity to restrict viral infection, however several family members are also capable of causing cellular DNA damage that leads to activation of DNA damage responses. Mutational signatures consistent with APOBEC3 activity have been identified in cancer genomes, and elevated expression of APOBEC3 enzymes has been observed in solid tumors. These findings point to a role for APOBEC3 enzymes in cancer-associated mutagenesis, however the impact of APOBEC3 activity within cancer cells has not been examined to date. We hypothesized that A3A is a potential source of mutagenesis in leukemia, since it is endogenously expressed at high levels in hematopoietic cells. To identify hematologic malignancies in which A3A expression is elevated, we examined RNA sequencing from adult and pediatric leukemias in the TCGA and TARGET databases. We found elevated A3A expression in a subset of pediatric and adult acute myeloid leukemia (AML). Additionally, we found that high A3A expression was associated with decreased survival, suggesting that new therapeutic options are important for patients with this type of malignancy. Since we have previously demonstrated that A3A acts on the cellular genome during replication, we hypothesized that abrogation of the replication checkpoint will produce genotoxicity and cell death in leukemias with elevated A3A expression. We therefore investigated the DNA damage response to A3A activity in leukemia cells by generating cell lines with inducible A3A expression. We found increased Ataxia Telangiectasia and Rad3-Related (ATR)-mediated phosphorylation of Chk1 (at serine 317 and serine 345) by Western blotting, and G2 arrest by flow cytometry. These results demonstrate that A3A causes replication checkpoint activation in leukemia cell lines. We investigated the potential for selective targeting of A3A-expressing AML cells by inhibition of replication checkpoint kinases. We found that A3A expression sensitized THP1 cells to ATR kinase inhibition, resulting in decreased viability, apoptosis, and abrogation of cell cycle arrest. We also found that Chk1 inhibition combined with A3A expression results in synthetic lethality in these cells. We determined that synthetic lethality with A3A is specific to the inhibition of ATR-Chk1 pathway and does not occur after inhibition of Ataxia Telangiectasia Mutated (ATM). We propose that A3A acts on ssDNA during replication in leukemia cells. ATR-Chk1 signaling is required for genome protection when A3A is active, and inhibition of this checkpoint results in cytotoxicity. These data reveal a new vulnerability of AML cells with elevated A3A expression and suggest that A3A is a biomarker for treatment with ATR kinase inhibitors. This abstract is also being presented as Poster B18. Citation Format: Abby M. Green, Konstantin Budagyan, Katharina E. Hayer, Matthew D. Weitzman. APOBEC3A sensitizes leukemia cells to inhibitors of the replication checkpoint [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR09.

Chemotherapy-Dependent ATP Release from Leukemia Dying Cells Induces Indoleamine 2,3-Dioxygenase 1 in Dendritic Cells

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) remains poor due to the lack of novel and effective therapies. The cancer cell death induced by some chemotherapeutic agents, especially anthracyclines, such as daunorubicin (DNR), named immunogenic cell death (ICD), is characterized by intra- and peri-cellular modifications, which favor the induction of anti-tumor T-cell immune response. Among them, the extracellular release of adenosine triphosphate (extracellular ATP, eATP) from dying tumor cells primes dendritic cells (DCs) by activating purinergic P2X7 receptors, thus eliciting the presentation of tumor antigens to T cell. DCs are key regulators of adaptive immunity, promoting or suppressing T-cell responses. One of the suppressive mechanisms involves the expression of indoleamine 2,3-dioxygenase 1 (IDO1), which plays a major role in the induction of T-cell tolerance through the expansion of regulatory T cells (Tregs). The present study aimed at evaluating the involvement of IDO-1 during ATP-driven ICD in AML.METHODS: AML patients were analyzed at diagnosis and after DNR-based chemotherapy. Ex vivo T cells were characterized by FACS and tested for their capacity to produce IFN-γ in response to autologous blasts. Then, CD8+IFN-γ-producing T cells were expanded and further characterized. ATP was used as an ICD representative model. In vitro, murine WEHI-3B and human HL-60 leukemic cell lines and primary blasts were tested for ATP release after DNR treatment. To in vivo investigate DNR-induced ICD, WEHI-3B cells stable transfected with luciferase PmeLUC were inoculated subcutaneously in BALB/c mice to measure ATP release directly from tumor mass. Tumor infiltrating DCs and T cells were characterized by FACS and immunohistochemistry after chemotherapy and plasma levels of cytokines were measured. In vitro DNR-treated AML cells were pulsed into immature DCs, previously generated from healthy donors. DCs maturation and IDO1 expression were examined (by FACS and western blot, respectively) and correlated with the presence of ATP in culture medium. IDO-driven Tregs induction was assessed. Finally, functional immunological tests were performed in vitro to test the ability of Tregs to inhibit leukemia antigen-specific IFN-γ production (FACS analysis) by ICD-activated T cells.RESULTS: After chemotherapy, 15/23 AML patients had an increase in leukemia-specific IFN-γ producing CD4+ and CD8+ T cells. Also an increase of Tregs was observed with a peak at day 21. CD8+ IFN-γ-producing T cells, which resulted in a skewing toward an effector memory phenotype, were activated and cytotoxic against autologous AML blasts but showed features of exhaustion and were defective in perforin production. In vitro and in vivo DNR induced ATP release from AML cells. In vivo the analysis of tumor-infiltrating T cells after treatment has shown an exhausted phenotype of cytotoxic CD8+ cells, increased IFN-γ+ Tregs and decreased TNF-α+ effector T cells. DNR treatment also increased in vivo plasma levels of cytokines IFN-γ, IL-1β, TNF-α, IL-12. Moreover, in DNR-treated mice we observed a significant increase of CD11c+ mature DCs which express IDO1 in tumor infiltrate. In vitro, loading of DNR-treated AML cells into DCs resulted in increased maturation, but also in IDO1 induction. Interestingly, extracellular ATP was directly involved in DCs maturation and IDO1 expression via purinergic receptor P2Y11. ICD-driven DCs were able to expand Tregs in an IDO-dependent manner. Finally, ICD both triggers a leukemia-specific IFN-γ production by CD8+T cells and induces Tregs, via IDO1-expressing DCs, which in turn inhibit leukemia-specific T cell.CONCLUSIONS: Overall, our data indicate that in AML chemotherapy-induced ICD has contrasting, and not fully elucidated, effects on T-cell immune response, resulting in the induction of leukemia-specific CTLs, albeit with defective features, and Tregs. In this scenario, the effects of ATP release from dying leukemia cells on DCs may be pivotal, as indicated by its capacity to concomitantly induce DC maturation and activation as well as tolerogenic function via IDO1. The combination of novel immunological drugs, such as IDO1 and/or checkpoint inhibitors, with conventional chemotherapy may represent an interesting approach to contrast tolerance induction and, then, fully exploit the immunogenic effect of chemotherapy. DisclosuresMartinelli:Genentech: Consultancy; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; MSD: Consultancy; BMS: Speakers Bureau. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Abstract 3766: Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs

Abstract Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that support tumor development and maintenance. In many instances, these oncogenic drivers are ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. We investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific oncogenic drivers, such as c-Myc and Bcl-2, used by tumor cells for growth and survival. We tested the activity of two GQIDs, GQC-05 and GSA-1103, on cell growth of a panel of eight pediatric and eight adult acute myeloid leukemia (AML) cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 μM for GQC-05 and from 40 nM to 2 μM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-Myc and/or Bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of the highly sensitive line MV-4-11 with GQC-05 showed a 3-4 fold decrease in expression of c-Myc and Bcl-2 mRNA. Furthermore, GQC-05 treatment resulted in decreased levels of both c-Myc and Bcl-2 proteins. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures. Citation Format: Megan Turnidge, Justin J. Montoya, Apurvi Patel, David W. Lee, Eiman Aleem, Daniel H. Wai, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs. [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 3766.

The Induction of Inhibitory Pathways in Dendritic Cells May Hamper the Efficient Activation of Anti-Leukemia T Cells within Chemotherapy-Induced Immunogenic Cell Death

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) is still poor due to the lack of novel and effective therapies. In different malignancies including AML, some chemotherapy agents, such as daunorubicin (DNR) but not cytarabine (Ara-C), activate the immune response via the cross-priming of anti-tumor T cells by dendritic cells (DCs). Such process, known as immunogenic cell death (ICD), is characterized by intracellular and pericellular modifications of tumor cells, such as the cell surface translocation of calreticulin (CRT) and heat shock proteins 70/90 (HSPs 70/90), the extracellular release of ATP and pro-inflammatory factor HMGB1. Alongside with ICD, chemotherapy is known to induce inflammatory modifications within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, which may ultimately affect anti-tumor T-cell responses. In this study, we characterize ICD in AML to evaluate the involvement of some DC-related inhibitory pathways, such as the expression of indoleamine-2,3-dioxygenase 1 (IDO1) and the activation of PD-L1/PD-1 axis.METHODS: AML patients were analyzed at diagnosis.Before and after DNR-based chemotherapy, patient-derived T cells were extensively characterized by FACS and analyzed for their capacity to produce IFN-γ in response to autologous blasts. The AML cell line HL-60 and primary AML cells were then exposed, in vitro, to different drugs, including DNR and, as control drug, Ara-C. Dying cells were tested for the surface expression of CRT and HSPs 70/90, the release of HMGB1 and ATP. Functionally, immature DCs generated from healthy donors were pulsed with DNR-treated AML cells. Then, loaded DCs were tested for the expression of maturation-associated markers and of inhibitory pathways, such as IDO1 and PD-L1 and used to stimulate autologous CD3+ T cells. After co-culture, autologous healthy donor T cells were analyzed for IFN-g production, PD-1 expression and Tregs induction. A mouse model was set up to investigate in vivo the mechanism(s) underlying ICD in AML. The murine myelomonocytic leukemia cell line WEHI was transfected with luciferase PmeLUC probe, inoculated subcutaneously into BALB/c mice and used to measure in vivo ATP release after chemotherapy. Tumor-infiltrating T cells and DCs were characterized and correlated with ATP release.RESULTS: DNR treatment induced ICD-related modifications in both AML cell lines and primary blasts, including CRT, HSP70 and HSP90 exposure on cell surface, HMGB1 release from nucleus to cytoplasm and supernatant increase of ATP. Ex vivo, T-cell monitoring of DNR-treated AML patients displayed an increase in leukemia-specific IFN-g-producing CD4+ and CD8+ T cells in 20/28 evaluated patients. However, FACS analysis of CD8+ effector T cells emerging after chemotherapy showed a significant up-regulation of exhaustion marker such as LAG3 and PD-1, which paralleled with their reduced ability to produce active effector molecules, such as perforin and granzyme. Moreover, an increase of circulating Tregs was observed after DNR-based chemotherapy. In vitro, loading of chemotherapy-treated AML cells into DCs resulted not only in the induction of a maturation phenotype, but also in over-expression of inhibitory pathways, such as IDO1 and PD-L1. The silencing of IDO1 increased the capacity of DCs loaded with DNR-treated AML cells to induce leukemia-specific IFN-γ production by CD4+ and CD8+ T cells. In vivo, DNR therapy of mice inoculated with established murine AML cell line resulted in increased ATP release. Similarly to ex vivo and in vitro results, tumor-infiltrating DCs showed an increase in maturation status. Moreover, CD4+ and CD8+ T cells had increased IFN-γ production, but showed an exhausted phenotype.CONCLUSIONS: Our data confirm that chemotherapy-induced ICD may be active in AML and results in increased leukemia-specific T-cell immune response. However, a deep, ex vivo, in vitro and in vivo characterization of chemotherapy-induced T cells demonstrated an exhausted phenotype, which may be the result of the inhibitory pathways induction in DCs, such as IDO and PD-L1. The present data suggest that combination of chemotherapy with inhibitors of IDO1 and PD-L1 may represent an interesting approach to potentiate the immunogenic effect of chemotherapy, thus resulting in increased anti-leukemia immune response. DisclosuresCavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

Preclinical Activity of a New Proteasome Inhibitor, MLN9708, in Acute Myelogenous Leukemias Expressing NPM1 Mutated Protein

ABSTRACT: The nucleophosmin-1 (NPM1) mutation represents the most common genetic lesion (30-35%) in adult acute myeloid leukemia (AML) with the mutation resulting in the cytoplasmic delocalization of the NPM protein (NPMc+, most commonly mutation type A, c.860_863dupTCTG). Outside of conventional chemotherapy, treatment options for those with relapsed disease are extremely limited. We have previously discovered that leukemic cells expressing NPMc+ are preferentially sensitive to the cytotoxic effects of the first generation proteasome inhibitor, bortezomib, as result of the induction of superoxide in these cells (Huang M, et al., Leukemia, 2013). However, bortezomib has demonstrated only modest anti-leukemic activity as a single agent in early phase clinical trials that have included primarily heavily pretreated patients not stratified according to mutational genotype. In addition, the high incidence of clinically significant peripheral neuropathy in these studies led to early discontinuation of treatment. MLN9708 (Ixazomib) is a novel second generation proteasome inhibitor that is orally bioavailable and has shown promising activity and improved tolerability in early phase clinical trials in lymphoma and in multiple myeloma, where it is currently in a Phase III clinical trial. The efficacy of MLN9708 in relapsed patients with AML has not yet been examined. We have investigated the in vitro effects of MLN9708 on the cytotoxicity, generation of reactive oxygen species (ROS), and cellular glutathione in both cultured NPMc+ AML cell lines and primary leukemic samples. Cultured OCI-AML3 cells (human AML cell line featuring the NPMc+ mutation) were approximately 50-fold more sensitive to MLN9708 with a mean half inhibitory concentrations (IC50 ± SD) of 67 nM ± 1.83 nM at 24 hours than were an AML cell line (THP-1) expressing wild type NPM1. Knockdown of both NPM1/NPMc+ by inducible NPM1 shRNA expression significantly attenuated the MLN9708-mediated cytotoxicity, as determined by MTS colorimetric and flow cytometric apoptosis assays. As with bortezomib, MLN9708 treatment at 75 nM induced a 2.1-fold (p < 0.002) increase in superoxide-specific DHE (PE)-derived fluorescence as compared to controls. Pre-incubation of cells with 25 mM N-acetylcysteine, a ROS scavenger, dramatically reduced cytotoxicity, strongly implicating the generation of ROS in the mechanism of MLN9708-induced cytotoxicity. Knockdown of NPM1/ NPMc+ expression resulted in an increase of glutathione levels of 2.6-fold (p < 0.04) in untreated cells, and 2.3-fold (p < 0.005) and 2.9-fold (p < 0.004) after a 22 hour incubation with MLN9708 at pharmacologically achievable doses of 75 nM and 150 nM, respectively (Figure 1). In addition, a concomitant 4-fold reduction (p < 0.04) in the NADP+/NADPH ratio was observed in the NPM1/NPMc+ depleted OCI-AML3 cells. In vitro apoptosis assays of primary leukemic blasts isolated from five patients with relapsed NPMc+ AML showed an IC50 for MLN9708 ranging from 49.963-467.487nM (183.8 nM ± 176.8 nM). Based on these promising in vitro studies, a Phase 2 clinical trial of single-agent oral MLN9708 (clinicaltrials.gov identifier: NCT02030405) has been initiated for patients with relapsed or refractory NPMc+ AML.We conclude that the expression of NPMc+ renders AML cells more susceptible to MLN9708 toxicity and that this effect results at least in part from the generation of superoxide. Lower glutathione levels in NPMc+ expressing cells suggests an underlying defect in the antioxidant defense pathway. Demonstration of the sensitivity of NPMc+ AML cells to MLN9708 as a single agent may serve as a platform for the development of novel combination strategies with this drug.Acknowledgments: Research was facilitated by the ASH Clinical Research Training Institute and AACR/ASCO Clinical Cancer Research training program. J.S.G is funded by the Leukemia & Lymphoma Society Career Development Award and ASCO YIA award through a grant supported by Mr. Aaron Sasson. J.S.G. is further supported by the Stanford Hematology Fellowship Program. [Display omitted] DisclosuresOff Label Use: MLN9708 is an investigational compound by Millennium Pharmaceuticals, Inc. (MPI). Through an MTA we have secured the compound for in vitro analysis. This data has led to a clinical trial and drug for that study is supplied by MPI.. Medeiros:Agios: Consulting - Ad board Other.

The Expression of Nrf2 in Adult Patients with De Novo Acute Myeloid Leukemia and Its Clinical Significance

Background: Nuclear factor erythroid-2 related factor-2(Nrf2), a helix-loop-helix basic leucine zipper transcription factor, is a key regulator in the cellular defense system against oxidative stress. Nrf2 is binding with Keap1 in a physiological manner, activation of Nrf2 by ROS results in the induction of a series of anti-oxidative stress/detoxifying enzymes and proteins, such as HO-1, NQO-1, UGT and GST. Previous studies suggest that, Nrf2 is aberrant activated in many cancer cells, which is playing an important role in the drug-resistance of the patients. What’s more, it was reported that there’s also an aberrant activation of Nrf2 in the AML cells, which leading to the chemotherapy resistance of AML patients. But the value of Nrf2 in prognosis of adult acute myeloid leukemia(AML) still remain unknown.Aims: The aim of this study was to determine Nrf2 expression in adult patients with de novo acute myeloid leukemia and its clinical significance.Methods: BM samples form 110 adult patients with de novo AML were analyzed. Of the 110 AML patients, 66 were males and 44 were females, with a mean age of 32 years (range from 12 to 77 years). Among them, 1 out of 110 patients was M1, 49 were M2, 14 were M4, 28 were M5, 1was M6 and 17 were acute unclassified leukemia. All patients received 1 to 2 cycles of induction of standard-dose cytarabine continuous infusion×7 days with idarubicin or daunorubicin×3days, fellowed by consolidation therapy with HiDAC and then stem cell transplantation according to patient’s condition. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of Nrf2 gene in 110 de novo adult AML patients, and the patients were divided into high and low Nrf2 expression groups accordint to the median expression of Nrf2 mRNA. Comparisons were performed using Mann-Whitney U test, Chi-square test and Kaplan-Meier method.Results:Distribution of Nrf2 gene expression in different FAB subtypes was with no significant differences (P=0.0.385). The median age of AML pateints in low and high Nrf2 gene epxression groups were 35 and 39 years old (P=0.385), and the median expression of Nrf2 in 44 female patients and 66 male patients was 0.034 and 0.030 separately (P=0.696). What’s more, there was no significant difference of WBC, Hb level, PLT, bone marrow blast counts between the two groups (P＞0.05), and the Nrf2 expression level was also not correlated with chromosome risk stratification and the expression of CD34 (P＞0.05). The patients in high Nrf2 expression group got the same complete remission (72.7%) with the low Nrf2 epxression group. The survival curve gave the clues that the median survival period of hig and low Nrf2 expression groups were 218d and 361d, the overall survival rate was also with no significant difference in the two groups(72.7% vs 80.0%，P=0.667).Conclusions: Our results showed that no matter the expression level of Nrf2 gene, there are no significant difference of CR rate and OS rate in the patients with de novo acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Clinical Significance of Additional Cytogenetic Abnormalities (ACA) Acquired at the First Relapse in Adult Acute Myeloid Leukemia (AML) Patients

Background: Cytogenetic changes between the time of diagnosis and the first relapse are found in 30 to 60% of relapsed AML cases. ACA, the most frequent form of cytogenetic changes, are considered as a result of genetic instability and clonal evolution of AML cells. However, because the clinical significance of ACA has not been elucidated in adult AML patients, we conducted a large-scale retrospective study to address this unsolved issue.Patients and methods: Of the 375 adult patients diagnosed with AML between 1990 and 2010, 144 relapsed patients whose cytogenetic abnormality data both at diagnosis and at the first relapse were available were included in this study. All these patients underwent intensive chemotherapy. Patients with acute promyelocytic leukemia were excluded. Cytogenetic changes between the time of diagnosis and the first relapse were classified into four groups: (1) no change, (2) ACA was acquired at time of the first relapse, (3) cytogenetic abnormalities observed at diagnosis were reduced or had disappeared at the time of the first relapse, and (4) cytogenetic abnormalities observed at diagnosis were reduced or had disappeared, and completely different ACA was acquired at the time of the first relapse. In this study, groups 2 and 4 were defined as those with ACA acquisition. Overall survival (OS) was defined as the interval from the date of the first relapse to the date of death. Fisher’s exact test was used to compare binary variables. OS was estimated with the Kaplan-Meier method and compared using the log-rank test. Multiple logistic regression analysis and the Cox proportional hazard model were used for multivariate analysis of predisposing factors and prognostic factors, respectively. Values of p < 0.05 were considered to indicate statistical significance.Results: Of the 144 patients included in this study, 81 patients were male, and 63 were female. The median age was 53 years (range, 15–79 years). According to the definition described above regarding cytogenetic changes between the time of diagnosis and the first relapse, 83 (58%), 55 (38%), 2 (1%), and 4 (3%) patients were categorized into group 1, 2, 3, and 4, respectively; thus, 59 patients (41%) acquired ACA at the first relapse. With univariate analysis, t(8;21), complex karyotype, fewer than 12 months duration of the first remission, and relapse after allogeneic stem cell transplantation (allo-SCT) were extracted as statistically significant predisposing factors for ACA acquisition at the first relapse. Of these four factors, the first three were confirmed with multivariate analysis to be independent predisposing factors. Excluding four patients that directly proceeded to allo-SCT without re-induction chemotherapy, the 140 patients with ACA acquisition showed a significantly lower second remission rate compared with those without ACA acquisition (20.0% vs. 72.5%, respectively; p < 0.001). Furthermore, among all 144 patients, the 3-year OS rate after the first relapse in patients with ACA acquisition was significantly poorer than that in those without ACA acquisition (8.5% vs. 36.8%, respectively; p < 0.001). Multivariate analysis revealed that ACA acquisition was the strongest negative prognostic factor compared to all other reported factors (hazard ratio: 2.13, p < 0.001). Of the 59 patients with ACA acquisition (median age: 51, range; 15–79 years), 27 underwent allo-SCT after the first relapse. Eight patients were in remission at the time of transplant, and one directly proceeded to allo-SCT without re-induction chemotherapy. The 3-year OS rates after the first relapse were significantly different between patients undergoing allo-SCT after the first relapse and those treated only with chemotherapy (17.8% vs. 0%; p = 0.007). With multivariate analysis, undergoing allo-SCT after the first relapse was identified as an independent prognostic factor, in addition to the cytogenetic risk group at diagnosis.Conclusion: These findings suggested that ACA acquisition at the time of the first relapse was associated with chemo-refractive disease and poor prognosis. As ACA acquisition showed the most potent prognostic impact, treatment approaches for relapsed AML patients should be determined with consideration of this phenomenon. Clarification of the biological background of ACA acquisition may contribute to the development of novel therapeutic strategies and improved treatment outcomes in adult AML patients. DisclosuresNo relevant conflicts of interest to declare.

Regulation of p21 by TWIST2 contributes to its tumor-suppressor function in human acute myeloid leukemia.

TWIST2 has a dual function in tumors. Its implication in the initiation and metastasis of various solid tumors is well established, and its tumor-suppressor role in murine osteosarcoma cells has been reported recently. However, the function of TWIST2 and its underlying mechanisms in human normal and malignant hematopoiesis remain unclear. In the present study, we found that TWIST2 directly regulated p21 in human hematopoietic cells and whose silence promoted cell proliferation and cell cycle progression. Hypermethylation of TWIST2 occurred to 23 out of the 75 adult acute myeloid leukemia (AML) patients and resulted in the impaired expression of both TWIST2 and p21. Conversely, TWIST2 overexpression inhibited the growth of AML cells partially through its direct activation of p21 with intact HLH (helix-loop-helix) domain. The microarray data and gene expression validation showed that TWIST2 was sufficient to activate known tumor-suppressor genes, whereas suppress known oncogenes, which further supported its inhibitory effect against AML cells. Taken together, our data have identified a novel TWIST2-p21 axis that modulates the cell cycle of both normal and leukemic cells and demonstrated that the direct regulation of p21 by TWIST2 has a role in its tumor-suppressor function in AML.

Abstract 5220: Identifying AML-specific key targeted drugs using high-throughput drug sensitivity and resistance testing profiles of AML cells.

Abstract Introduction: Conventional cytotoxic chemotherapy regimens for adult acute myeloid leukemia (AML) are effective in curing less than 50% of the patients, and there is a major need for targeted drugs with better anti-cancer selectivity. Here, our aim was to i) identify potential clinically used or emerging cancer drugs by quantitative drug sensitivity and resistance testing (DSRT) of 16 AML cell lines ii) compare the cell line data with results obtained from tested 24 ex vivo AML patient specimens iii) identify genomic correlations potentially explaining drug responsiveness. Methods: The cancer pharmacopeia-wide drug collection is composed of 119 FDA approved and 90 investigational chemical compounds including cytotoxic agents and cell signaling molecule inhibitors. Each drug was tested over a 10,000-fold concentration range and that has generated quantitative five point dose-response curves. AML cells were plated in 384 well plates (where the drugs were pre-printed using an acoustic nano-dispensing technology, Labcyte®) and incubated in standard cell culture conditions. Cell viability was measured by Cell Titer Glow® luminescence assays. Analysis of dose response curves using Dotmatics® software resulted in IC50 values. Moreover, the genomic profiles of the AML cell lines were determined by microarrays and/or next-gen sequencing data for further integration with drug responses. Results: Comprehensive data analysis of 16 AML cell lines indicated that specific targeted drugs were selectively killing AML cells. The data analysis revealed relatively strong responses for MEK inhibitors in most AML cell lines (e.g. refametinib 87%, trametinib 82%, selumetinib 75%) while 21% of ex vivo AML patient samples were sensitive to these MEK inhibitors. In case of rapalog sensitivity, 80% of AML cell lines (e.g. temsirolimus 82%, everolimus 71%, sirolimus 81%) and 25% of ex vivo AML patient cases were responsive to the mTOR inhibitors. The AML cell lines carrying FLT3-ITD mutations were extremely sensitive to FLT3 inhibitors (e.g. quizartinib, lestaurtinib, tandutinib, and sorafenib) but very few responses to FLT3 inhibitors were observed in AML patients carrying an ITD mutation in the FLT3 kinase. Summary: Systematic DSRT profiling of AML cell lines illustrates drug sensitivity patterns to classify the cell lines as sensitive or resistant to specific classes of drugs. mTOR and MEK inhibitors were among the most effective inhibitors for most cell lines and also in some ex vivo patient cases suggesting that these drugs may have potential as therapeutic agents in AML. Also, bioinformatics predictions can be used to identify key synergistic combinations of tested drugs for effective AML therapy. Further integration of molecular profiles and functional responses of AML cell lines will help provide better understanding of drug efficacy based on known genetic background of the disease. Citation Format: Disha Malani, Astrid Murumägi, Tea Pemovska, Bhagwan Yadav, Evgeny Kulesskiy, Jing Tang, John Patrick Mpindi, Maija Wolf, Riikka Karjalainen, Tero Aittokallio, Caroline Heckman, Kimmo Porkka, Krister Wennerberg, Olli Kallioniemi. Identifying AML-specific key targeted drugs using high-throughput drug sensitivity and resistance testing profiles of AML cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5220. doi:10.1158/1538-7445.AM2013-5220