Chemotherapy-resistant Leukemic Stem Cells Research Articles

Abstract B174: The non-covalent HCK/BTK inhibitor TTX-114 displays potent in vitro and in vivo anti-tumor activity in leukemia and lymphoma

Abstract Background: The SRC family kinase Hematopoietic Cell Kinase (HCK) is upregulated in cycle–quiescent and chemotherapy-resistant leukemic stem cells (LSCs), and inhibition of HCK reduces the survival and self-renewal capacity of LSCs. As such, targeting HCK has emerged as a potential therapeutic strategy in acute myeloid leukemia (AML). In lymphoma, HCK phosphorylates and activates Bruton Tyrosine Kinase (BTK) downstream of Toll-like receptor (TLR) or the IL6 receptor. In conjunction with BTK, HCK engages essential pro-survival signaling through the NF-kB, mTOR, AKT and STAT pathways. Meanwhile, in lymphoma, HCK can take over pro-survival signaling to confer resistance to BTK inhibition. A dual HCK/BTK inhibitor thus provides a promising therapeutic approach to target leukemias and lymphomas. Methods: TTX-114 was identified through a structure-based medicinal chemistry campaign and characterized in cells using Western Blot and Cell Titer Glow for signaling and viability read-outs, respectively. For in vivo efficacy analysis, NSG newborn mice were irradiated prior to bone-marrow reconstitution with patient-derived primary AML LCSs. Mice were treated orally with TTX-114 after AML was established. Results: Here, we describe TTX-114, a non-covalent HCK/BTK inhibitor with anti-tumor activity in leukemia and lymphoma. In biochemical kinase profiling TTX-114 displayed sub-nM activity against BTK with a KD = 0.58 nM and the SRC-kinase cluster with a KD = 0.23nM for HCK. By inhibiting HCK and BTK activity in cells, TTX-114 prevented autophosphorylation of HCK at Y411 and BTK at Y223 in a dose-dependent manner in human peripheral blood mononuclear cells (PBMCs), enabling a starting point for clinical demonstration of target engagement. TTX-114 had anti-tumor activity across a larger panel of leukemia and lymphoma cell lines, with diffuse large B cell lymphoma (DLBCL) cell lines being among the most sensitive (IC50, ≤4 nM). In marginal zone lymphoma (MZL), the activity was slightly higher in the presence of MYD88 L265P mutation. In vivo, TTX-114 treatment significantly reduced the tumor burden in a dose and time-dependent manner in AML-derived PDX mice. A systematic pharmacokinetic/pharmacodynamic (PK/PD) study identified distinct PK driver for efficacy and tolerability, respectively enabling a well-tolerated dosing strategy for TTX-114 which induced complete response in AML-xenograft bearing mice with no impact on body weight. Conclusion: TTX-114 is an orally bioavailable, non-covalent HCK/BTK inhibitor which prevents HCK over-activation in cancer cells and provides a new targeted therapy for leukemias and lymphomas. Citation Format: Ulrike Rauh, Michael Serrano-Wu, Virendar Kaushik, Sabine Pilari, Ikuko Ogahara, Akiko Kaneko, Alberto J Arribas, Eleonora Cannas, Franceso Bertoni, Nathalie R Javidi-Sharifi, Matthew S Davids, Takehiro Fukami, Yoriko Saito, Fumihiko Ishikawa, Anthony Letai, Brian Hubbard. The non-covalent HCK/BTK inhibitor TTX-114 displays potent in vitro and in vivo anti-tumor activity in leukemia and lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B174.

Selective homing of CAR-CIK cells to the bone marrow niche enhances control of the acute myeloid leukemia burden

AbstractAcute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML is still unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)–T-cell therapy in AML has been hampered by several factors, including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche in which chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within this niche, could improve T-cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells, facilitating disease eradication. Specifically, we engineered conventional CD33.CAR–cytokine-induced killer cells (CIKs) with the wild-type (wt) CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of patients with warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndrome. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell–conditioned medium, with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs coexpressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a noncanonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.

Spatially Confined T-Cell Mediated Anti-AML Tumor Immunity By Dual Recognition Antibodies (CD123/CD200)

Definitive cure of acute myeloid leukemia (AML) remains an unmet need. While recombinant antibody-based and CAR-T cell mediated immunotherapies bear great potential in targeting chemotherapy-resistant leukemic stem cells, these approaches are largely limited by the lack of specific targets uniquely expressed by leukemic cells. Here, we propose a combinatorial strategy using a two-target approach which may have improved on-target and reduced off-target effects. The well-known stem cell marker interleukin-3 receptor α (CD123) is expressed on most AML cells. CD200 recently emerged as a cell surface marker mediating immuno-evasion on high-risk AML cells. We engineered the "Prodrug-activating chain Exchange"(PACE) TriFAB system (Dickopf S et al. Biological Chemistry, 2022) to yield a combinatorial anti-AML directed T-cell response. PACE-TriFAB educts are complementary Knob-into-hole IgG derived prodrugs in which one half is exchanged by a dummy containing an interface destabilizing mutation in the CH3 domain. The CH2 domain of the main body is replaced by a CD3 FAB light or heavy chain, respectively. With the hinge region being replaced by a flexible linker and therefore lacking disulfide bonds, a chain exchange between two educts is enabled when in close proximity on the cell surface. This leads to an activated anti CD3 domain and a dummy-dummy by-product. To demonstrate the T-cell activating potential, we co-cultured fresh PBMCs from healthy donors together with the CD200+/CD123+ AML cell line Kasumi-1 and checked T-cell activation by flow cytometry (E:T ratio 5:1, prodrug conc. 20 nM, 24 h stimulation). We observed a marked increase in CD69, CD25 and CD137 expression on CD8+ cells in the case when the CD123 and CD200 prodrugs were applicated simultaneously together with target cells, but not when single prodrugs alone or without target cells were analyzed (Median fluorescence intensity for CD25, CD69 and CD137: No construct: 450, 8.900 & 2.100; prodrug CD200: 460, 8.700 & 2.100; prodrug CD123: 460, 9.600 & 2.000; both prodrugs: 6.500, 344.300 & 43.100; results confirmed in repeat experiments) For the evaluation of the specific cytotoxic effect of the construct we transduced Kasumi-1 cells with an mCherry lentiviral vector and imaged the cells co-cultured with PBMCs every 2 h over a time span of 60 h (E:T ratio 5:1, prodrug conc. 10 nM). Proliferation of the Kasumi-1-mCherry cells was observed when no prodrug or only a single prodrug was added. However, application of both prodrugs together led to a decrease in mCherry positive cell count (log2 fold changes to starting point: No PBMCs 1,2; no construct 0,9; prodrug CD200 0,9; prodrug CD123 0,4; both prodrugs -2,1; n=3). Not only could we show that this elimination of tumor cells was dose dependent but also it was constrained by the E:T ratio (pivot point from killing to proliferation at 0,1 nM and E:T of 1:2 n=3). Similar results were obtained using patient derived xenograft (PDX) AML cells. In contrast to Kasumi-1 AML cells, specific killing of patient-derived PDX cells could be documented with lower E:T ratios and lower prodrug concentrations (pivot point from killing to proliferation at 0,05 nM and E:T of 1:5) We also started to analyze the anti-leukemic effect in vivo. Luciferase-expressing CD200+/CD123+ AML PDX cells were injected into NSG mice. On day +15, PBMC from a healthy individual were transplanted, followed by additional applications of the antibodies on days +16, +18, +21, +23, +25 & +29. Tumor burden was analyzed by bioluminescence imaging (log10 fold change [P/sec] 2 weeks after PBMC injection: Control 1,9; treated -0,4; group size = 3). In conclusion the combination of anti CD200 and anti CD123 as prodrugs of an on-target activating trispecific T-cell engager kills AML cells in vitro and in vivo and has the potential to eliminate AML LSC while minimizing the risk for severe on-target off-leukemia toxicity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Quantitative proteomics reveals specific metabolic features of acute myeloid leukemia stem cells

AbstractAcute myeloid leukemia is characterized by the accumulation of clonal myeloid blast cells unable to differentiate into mature leukocytes. Chemotherapy induces remission in the majority of patients, but relapse rates are high and lead to poor clinical outcomes. Because this is primarily caused by chemotherapy-resistant leukemic stem cells (LSCs), it is essential to eradicate LSCs to improve patient survival. LSCs have predominantly been studied at the transcript level, thus information about posttranscriptionally regulated genes and associated networks is lacking. Here, we extend our previous report on LSC proteomes to healthy age-matched hematopoietic stem and progenitor cells (HSPCs) and correlate the proteomes to the corresponding transcriptomes. By comparing LSCs to leukemic blasts and healthy HSPCs, we validate candidate LSC markers and highlight novel and potentially targetable proteins that are absent or only lowly expressed in HSPCs. In addition, our data provide strong evidence that LSCs harbor a characteristic energy metabolism, adhesion molecule composition, as well as RNA-processing properties. Furthermore, correlating proteome and transcript data of the same individual samples highlights the strength of proteome analyses, which are particularly potent in detecting alterations in metabolic pathways. In summary, our study provides a comprehensive proteomic and transcriptomic characterization of functionally validated LSCs, blasts, and healthy HSPCs, representing a valuable resource helping to design LSC-directed therapies.

Evaluation of a Bifunctional Sirpα-CD123 Fusion Antibody for the Elimination of Acute Myeloid Leukemia Stem Cells

Background: Despite considerable advances in the development of novel strategies for the treatment of acute myeloid leukemia (AML) the relapse rate is still high with only limited treatment options. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate outgrowth of the disease, highlighting the need of targeting LSCs to improve overall survival. Immunotherapies represent a promising strategy to target chemotherapy-resistant LSCs in AML. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows only a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen (Haubner et al, Leukemia 2019). CD47, known as a marker of self, is also highly expressed on LSCs as immune escape mechanism. CD47 transmits a "don't eat me" signal upon its interaction with the myeloid-specific signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs and provide AML patients a possibility for prolonged relapse-free survival, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123 positive AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity. Methods: The bifunctional SIRPα-CD123 antibody was generated by fusing the endogenous extracellular domain of SIRPα, which functions as the CD47 blocking domain, to an CD123 antibody CD123. We assessed the selective binding of the bifunctional antibody to CD123+CD47+ AML-derived cells and the ability to block CD47 on CD123+ cells in vitro. Furthermore, the biological activity of the SIRPα-CD123 antibody was examined using the AML-derived cell line MOLM-13, patient-derived xenografted (PDX) AML cells as well as primary cells from patients with newly diagnosed or relapsed AML. Results: We engrafted the endogenous SIRPα V-like domain to an antibody targeting CD123, which improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody (MFI ratioCD123 = 2.46 0.25 vs MFI ratioSIRPα-CD123 = 4.44 0.60). The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity (EC50CD123 = 38.5 pM vs EC50SIRPα-CD123 = 10.1 pM, n = 9). Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells ex vivo. Further, an improved ex vivo cytotoxicity towards AML PDX cells was observed with the SIRPα-CD123 antibody (% lysis at 100 nM: 14.27 5.40 vs 42.94 10.21 for CD123 and SIRPα-CD123 antibodies respectively, n = 3). With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ positive cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in allogenic as well as autologous settings (% phagocytosis, normalized to isotype control and maximum phagocytosis in an autologous setting: 20.11 4.59 vs 90.37 6.22, n = 5 for CD123 and SIRPα-CD123 antibodies, respectively). We were further able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity. Taken together, our in vitro data supports the elimination of the CD123+CD47+ positive AML LSC compartment by a synergistic effect of avidity-dependent binding to CD123 and CD47 and the simultaneous inhibition of the innate immune CD47-SIRPα signaling pathway. Conclusions: The SIRPα-CD123 is a bifunctional antibody with the potential to deplete CD123+CD47+ AML LSCs by a dual mode of action mechanism resulting in NK cell dependent cytotoxicity and macrophage-mediated phagocytosis. By combining a high affinity binding to CD123+ cells and a low affinity CD47 blockade that is restricted to CD123+ cancer cells we effectively minimize the risk for CD47-related on-target off-leukemia toxicity. The results of our in vitro assays using AML cell lines are consistent with the data from PDX and primary AML samples and support further preclinical testing of the SIRPα-CD123 antibody in vivo. Disclosures Subklewe: Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Oxford Biotherapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Janssen: Consultancy.

GRB10 IS A TUMOR SUPPRESSOR IN ACUTE MYELOID LEUKEMIA

Acute myeloid leukemia (AML) represents a major therapeutic challenge with high relapse rates due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs). To eradicate AML, a better understanding of the mechanisms that regulate LSC survival and resistance is essential. The PI3K/mTOR signaling pathway controls key signaling factors that promote the expansion and survival of chemotherapy-resistant LSCs. The mTOR complex 1 is negatively regulated by the imprinted gene, growth factor receptor-bound protein-10 (Grb10). In human AML, low Grb10 levels correlates with adverse prognosis and increased relapse rate. To study the role of Grb10 in regulating AML in vivo, we developed a HoxA9-Meis1 and MLL-AF9 driven murine AML models with maternal deletion of Grb10 (Grb10m/+). Primary and secondary recipient mice transplanted with Grb10m/+ leukemia cells displayed decreased survival (p

Absence of NKG2D Ligands Defines Human Acute Myeloid Leukaemia Stem Cells and Mediates Their Immune Evasion

Patients with acute myeloid leukaemia (AML) often achieve remission but subsequently die of relapse driven by chemotherapy resistant leukemic stem cells (LSCs). To initiate and maintain cancer, LSCs must also escape immunosurveillance. However, in vivo studies on human LSCs largely disregard lymphocyte mediated anti-tumor immunity due to the use of immunocompromised mice.Here we investigate the immunosurveillance mediated by NKG2D, a danger detector expressed by cytotoxic lymphocytes such as natural killer (NK) cells that recognizes stress-induced ligands (NKG2DL) of the MIC and ULBP protein families on AML cells. Staining of n=175 de novo AML with antibodies against MICA, MICB and ULB2/5/6 or an NKG2D-Fc chimeric protein recognizing pan-NKG2DL expression revealed NKG2DL to heterogeneously express among leukemic cells of the same patient (Fig. 1a). As expected, NKG2DLpos AML cells were efficiently cleared by natural killer (NK) cells, while NKG2DLneg leukemic cells escaped NK cell lysis. Interestingly, these NKG2DLneg AML cells also showed immature morphology, enhanced in vitro clonogenicity (39±47 colonies vs. 1±4, p<0.001, n=32 AML cases) and selective abilities to initiate leukemia in NSG mice devoid of functional NK cells (NKG2DLneg, 33/35, 94%; NKG2DLpos, 0/35, 0%; p<0.001, n=13 AML cases, Fig. 1b) and to survive chemotherapy in vivo. In mice, NKG2DLneg AML cells generated both NKG2DLpos and NKG2DLneg progeny of which again only latter induced leukemia in re-transplant assays. Even though similar leukemia-specific mutations were retrieved in NKG2DLneg and NKG2DLpos AML cells derived from the same patient (n=12 analysed patients), published LSC, HSC and 17-genes stemness score signatures were specifically enriched in NKG2DLneg fractions.Mechanistically, expression of poly-ADP-ribose polymerase 1 (PARP1) was identified as enriched in NKG2DLneg compared to NKG2DLpos leukemic subpopulations, and PARP1 inhibition (PARPi) using either siRNAs or pharmacological inhibitors such as AG-14361, veliparib, talazoparib or olaparib, increased NKG2DL mRNA transcripts between 6 and >50 fold. PARP1 binding sites were identified by in silico analysis in NKG2DL promoters and binding was confirmed by chromatin immunoprecipitation in the promoters of MICA and MICB. Importantly, treatment with PARPi induced NKG2DL surface expression on LSCs in vitro and in vivo and co-treatment with PARPi and NK cells efficiently suppressed leukemogenesis in patient derived xenograft (PDX) models (Fig. 1c). These data suggest that PARP1 inhibition sensitizes LSCs to NK cell mediated elimination.Finally, NKG2DL surface expression was found to inversely correlate with favorable molecular AML characteristics (favorable ELN risk group vs. other: p=0.034; inv(16) versus other: p=0.023), complete remission rates after induction chemotherapy (all patients: p=0.002, patients <65 years: p=0.004) and patient overall survival (patients <65 years: p=0.028). Enhanced PARP1 expression in leukemic cells furthermore associated with poor clinical outcome (TCGA data set, p=0.0038).In summary, our data link the concept of LSCs to immune escape in human AML and propose the absence of immunostimulatory NKG2DL as a novel method to identify LSCs across genetic AML subtypes (including CD34 negative AMLs). This LSC specific mechanism of immune evasion could be overcome by treatment with PARP1 inhibitors, which in conjunction with functional NK cells holds promise to eradicate LSCs and promote immune-mediated cure of AML.Fig. 1: Human AML contain NKG2DLpos as well as NKG2DLneg subpopulations but only latter display leukemia initiation capacity (a: left, analysis of n=175 AML cases using NKG2D-Fc staining, right: exemplary flow cytometry plots; b: leukemic infiltration and survival in mice transplanted with NKG2DLneg or NKG2DLpos AML cells sorted from the same AML cases). PARP1 inhibition with AG-14361 up-regulates NKG2DL on CD34+ LSCs, and in vivo co-treatment with AG-14361 and polyclonal allogeneic NK cells suppresses leukemogenesis in PDX models (c). DisclosuresSalih:Several patent applications: Patents & Royalties: e.g. EP3064507A1.

Pharmacologic Targeting of LIN28/Let-7 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy with high relapse rates and mortality due to the outgrowth of chemotherapy-resistant leukemic stem cells (LSCs). Thus, the development of novel therapeutic strategies capable of eradicating human AML represents a major area of unmet medical need. The RNA binding protein, LIN28, is a known driver of many cancer stem cells, AML included, wherein overexpression of LIN28 correlates with reduced patient survival. LIN28 blocks the function of the let-7 microRNA family, which exert tumor suppressive effects by repressing oncogenes and cell cycle regulators including MYC, RAS and CyclinD. Thus, LIN28 is an attractive mechanistic target for the purpose of inhibiting AML LSCs. Using a targeted high-throughput screen, we identified a class of small molecules which selectively block the LIN28/let-7 interaction (Roos et al., ACS Chem Biol, 2016). Preliminary studies demonstrate that a lead small molecule markedly impairs the proliferation and clonogenic capacity of human AML cell lines and primary patient AML samples. In vivo, systemic administration of a lead small molecule LIN28/let-7 inhibitor decreases leukemic tumor burden, reduces LSC numbers and significantly improves animal survival. Mechanistic studies revealed that targeted inhibition of the LIN28/let-7 axis restores let-7 microRNA levels in AML LSCs and subsequently inhibits a panoply of key oncogenic driver genes, including the NF-ĸB pathway, a hallmark for LSC proliferation. Furthermore, AML cell lines and primary patient cells treated with the LIN28/let-7 small molecule inhibitor showed a block at the G1/S phase interface and significantly decreased cell cycle progression. Consequently, LIN28/let-7 inhibition leads to LSC differentiation and ultimately leukemic cell death. In summary, we demonstrate for the first time the drugability of the LIN28/let-7 axis in vivo and reveal a novel pharmacological means to suppress a multitude of oncogenic driver genes in human AML. These results suggest that small molecule inhibition of LIN28/let-7 has high therapeutic potential as a new class of targeted therapies for AML. DisclosuresNo relevant conflicts of interest to declare.

CD25 targeted therapy of chemotherapy resistant leukemic stem cells using DR5 specific TRAIL peptide

Chemotherapy resistant leukemic stem cells (LSCs) are being targeted as a modern therapeutic approach to prevent disease relapse. LSCs isolated from methotrexate resistant side population (SP) of leukemic cell lines HL60 and MOLT4 exhibited high levels of CD25 and TRAIL R2/DR5 which are potential targets. Recombinant immunotoxin conjugating IL2α with TRAIL peptide mimetic was constructed for DR5 receptor specific targeting of LSCs and were tested in total cell population and LSCs. IL2-TRAIL peptide induced apoptosis in drug resistant SP cells from cell lines and showed potent cytotoxicity in PBMCs derived from leukemic patients with an efficacy of 81.25% in AML and 100% in CML, ALL and CLL. IL2-TRAIL peptide showed cytotoxicity in relapsed patient samples and was more effective than TRAIL or IL2-TRAIL proteins. Additionally, DR5 specific IL2-TRAIL peptide was effective in targeting and killing LSCs purified from cell lines [IC50: 952nM in HL60, 714nM in MOLT4] and relapsed patient blood samples with higher efficacy (85%) than IL2-TRAIL protein (46%). Hence, CD25 and DR5 specific targeting by IL2-TRAIL peptide may be an effective strategy for targeting drug resistant leukemic cells and LSCs.

Flow Cytometry Quantification of Leukemic Stem Cells Is Associated with Risk Stratification and May Be Useful for Minimal Residual Disease in Acute Myeloid Leukemia

Abstract 1470Investigation of minimal residual disease (MRD) after remission induction (RI) therapy provides important information for risk assessment in patients with acute myeloid leukemia (AML). The presence of immature and chemotherapy-resistant leukemic stem cells (LSC) within the bulk of AML blasts at diagnosis and in post-induction bone marrow (BM) may lead to relapses. Nevertheless, whether the frequency of cells with LSC characteristics and their clearance after induction is correlated with prognosis has not been established. Using four-color multiparametric flow cytometry (MFC), BM quantification of leukemic associated phenotypes (LAPs) and LSCs was performed at diagnosis and after the first RI from 37 AML patients, excluding acute promyelocytic leukemia, with a median age of 48 years and a male/female ratio of 0.95. All patients received the conventional daunorrubicin and cytarabin (3 + 7) RI chemotherapy. Complete remission (CR) was defined as BM blast count inferior to 5%. Thirty-three patients were classified according to the European Leukemia Net recommendation and 9/32 (28.1%) patients were allocated in favorable, 14/32 (43.8%) in intermediate and 9/32 (28.1%) in poor cytogenetic/molecular risk group. FLT3-ITD mutation was detected in 9/33 (27.3%) and 5/28 (17.9%) carried NPM1 mutation. MRD identification was performed on 20 erythrocyte-lysed whole BM samples after staining with a panel of directly conjugated monoclonal antibodies. Five patients were excluded from this analysis because a LAP could not be identified. Blasts gating was performed considering the low expression of CD45 and sideward scatter (SSC) and CD34 expression (these latter, when more than 20% of blasts were detected at diagnosis). Within this population, the following LAPs were investigated: CD15/CD117, HLA-DR/CD13, HLA-DR/CD33, CD2/CD56, CD19/CD11b, CD42a/CD33, CD64/CD11c or CD14/CD11c. LSCs were selected by the same CD45dim × SSC gating strategy and defined as CD34+/CD38−/CD123+. After staining procedures, at least one hundred thousand events were acquired in a FACScalibur flow cytometer and analysis was performed using the Cell Quest software. LAP and LSC quantification, at diagnosis and at days 21 to 30 of RI, was analyzed as a categorical variable defined as lower or higher than the median and was compared to the following variables: age (< or > 60 years old); WBC count (< or > 30 × 103); cytogenetic/molecular risk; and morphological CR. The comparison between LAP and LSC quantification at both time points was also assessed. Comparison of categorical variables was performed using Fisher’s exact test or Yates’ corrected chi-square for two or more variables, respectively. Statistical analyses were performed using SPSS 13.0 software and P < 0.05 was considered to be significant. LSC quantification at diagnosis was found at varying frequencies across different cytogenetic/molecular risk groups, being higher at the poor risk group (P = 0.041). Of note, 100% of the poor risk patients had high levels of LSC at diagnosis. In addition, the presence of FLT3-ITD mutation was associated with higher amounts of the LSC population at diagnosis (P = 0.043). The most frequent detected LAP was CD45dim/CD34+/HLA-DR+/CD13+. Low or high expression of the LAP was not correlated with the prognostic variables at diagnosis. CR rate was 83.33% and was not different in the groups with high or low levels of LSC at diagnosis. However, LAP and LSC quantification after RI were found to be correlated (P = 0.018), suggesting that the LSC subpopulation can be useful for MRD monitoring at this early treatment time-point. Therefore, LSC quantification by MFC at diagnosis can identify patients at high risk of relapse and offers the opportunity to study the stem cell compartment after chemotherapy. These findings are particularly important for the intermediate normal karyotype risk group patients, who frequently do not have specific molecular targets for MRD monitoring. Disclosures:No relevant conflicts of interest to declare.

High Expression of MDR1 and BCRP1 in Leukemic Stem Cells Correlates with Reversible Drug Resistance Ex Vivo and Predicts Failure of Remission Induction Chemotherapy in Patients with Acute Myeloid Leukemia.

Although overexpression of members of the ATP-Binding-Cassette (ABC) transporter superfamily has been implicated in chemotherapy failure in acute myeloid leukemia (AML) in some studies, results have been conflicting. Previously we found no consistent difference in pre-treatment ABC transporter expression when comparing total blasts of 18 patients achieving complete remission (CR) and 13 patients who were refractory to induction chemotherapy (NR). However, among the CD34+CD38− AML cells (enriched for candidate “leukemic stem cells” which engraft in immunodeficient mice), as assessed by quantitative RT-PCR, elevated expression of MDR1 and/or BCRP1, two ABC transporters associated with drug resistance, was found in 8/10 NR patients as compared to 0/7 CR patients. No difference between CR and NR samples was observed in the more differentiated CD34+CD38+ or CD34− AML cells while none of the subpopulations showed a significant difference in MRP1 expression between CR and NR patient samples. To determine if this difference in gene expression was associated with altered functional activity of MDR1/BCRP1, the relative sensitivity of subpopulations of AML cells to induction of apoptosis following ex vivo treatment with daunorubicin with or without ABC inhibitors (PSC-833 and Verapamil) was assessed. All subpopulations of AML cells isolated from 2 CR patients showed similar drug sensitivity profiles (IC50 <0.04 μg/ml). In contrast, CD34+CD38− cells from 6 NR patients were more resistant to daunorubicin-induced apoptosis (p<0.05 for comparison of IC50s between CR and NR samples). ABC transporter inhibition in CD34+CD38− cells did not change daunorubicin sensitivity in CR samples, but increased drug sensitivity in a dose-dependent manner in NR samples, particularly in the CD34+CD38− fraction. This suggests a role for ABC transporters in sustaining a population of chemotherapy resistant leukemic stem cells which may allow survival of the malignant clone in patients following induction therapy. If the value of measuring MDR1/BCRP1 expression among these cells in predicting chemotherapy outcome is confirmed in a larger patient population, it may be possible to use such analysis to modify therapy for individual AML patients to overcome drug resistance.