AML Blast Cells Research Articles

Immortalised chronic myeloid leukemia (CML) derived mesenchymal stromal cells (MSCs) line retains the immunomodulatory and chemoprotective properties of CML patient-derived MSCs

Despite the success of Tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML), leukemic stem cells (LSCs) persist, contributing to relapse and resistance. CML Mesenchymal Stromal Cells (MSCs) help in LSC maintenance and protection from TKIs. However, the limited passage and self-differentiation abilities of primary CML MSCs hinder extensive research. To overcome this, we generated and characterized an immortalised CML patient-derived MSC (iCML MSC) line and assessed its role in LSC maintenance. We also compared the immunophenotype and differentiation potential between primary CML MSCs at diagnosis, post-treatment, and with normal bone marrow MSCs. Notably, CML MSCs exhibited enhanced chondrogenic differentiation potential compared to normal MSCs. The iCML MSC line retained the trilineage differentiation potential and was genetically stable, enabling long-term investigations. Functional studies demonstrated that iCML MSCs protected CML CD34+ cells from imatinib-induced apoptosis, recapitulating the bone marrow microenvironment-mediated resistance observed in patients. iCML MSC-conditioned media enabled CML CD34+ and AML blast cells to proliferate rapidly, with no impact on healthy donor CD34+ cells. Gene expression profiling revealed dysregulated genes associated with calcium metabolism in CML CD34+ cells cocultured with iCML MSCs, providing insights into potential therapeutic targets. Further, cytokine profiling revealed that the primary CML MSC lines abundantly secreted 25 cytokines involved in immune regulation, supporting the hypothesis that CML MSCs create an immune modulatory microenvironment that promotes growth and protects against TKIs. Our study establishes the utility of iCML MSCs as a valuable model to investigate leukemic-stromal interactions and study candidate genes involved in mediating TKI resistance in CML LSCs.

Sgr-2921, a Potent CDC7 Inhibitor, Demonstrates Significant Anti-Leukemic Responses in Patient-Derived AML Models Representing Difficult-to-Treat Disease

Introduction: Elevated replication stress (RS) is a common feature of many highly proliferative cancers including AML. CDC7 has been shown to activate key components of the RS response, including ATR, and the BRCA1 and cohesin complexes, thereby mitigating accumulation of DNA damage and genomic instability. Inhibition of CDC7 in cancer cells results in an impaired response to RS, leading to accumulation of DNA damage and cell death. SGR-2921 is a potent inhibitor of CDC7 and a clinical trial to evaluate the safety, tolerability and preliminary antitumor activity of SGR-2921 in patients with AML and MDS is expected to begin in 2023 (NCT 05961839). We have previously shown that SGR-2921 has potent anti-proliferative activity in 12 out of 16 patient-derived AML samples ex vivo. Importantly, 6 out of 6 AML patient samples with mutations in TP53 responded to SGR-2921. Here we show that SGR-2921 results in potent, dose-dependent, anti-leukemic activity in multiple disseminated in vivo AML patient-derived xenograft (PDX) models representing difficult-to-treat disease. Methods: NOG-EXL mice were inoculated with primary human AML blast cells after sublethal irradiation. Engraftment of human AML blast cells were measured in surrogate mice by flow cytometry using human CD45 vs. mouse CD45 markers. When human CD45 engraftment reached >20% of bone marrow cells in surrogate mice, the study mice were randomized based on weight and dosed with vehicle, SGR-2921 at 3 dose levels or a standard of care agent. SGR-2921 was administered via oral gavage (PO) twice daily in cycles of 5 days of dosing followed by 9 days of no compound dosing over 2 cycles. In a separate study, phosphorylated MCM2 (a direct substrate of CDC7) was measured in the same cohort of mice engrafted with human AML cells following 3 doses of SGR-2921 to determine target engagement. Results: In vivo, SGR-2921 showed strong anti-leukemic activity in the AML PDX models at tolerated doses. Specifically, engrafted AML cells derived from a patient with TP53 mutated AML, a relapsed and refractory patient and a patient with KTM2a rearrangements showed significant and dose-dependent reduction in human CD45 positive AML blast cells. The reduction of blast cells was observed in both peripheral blood and bone marrow. The reduction in human CD45 positive blast cells were associated with an increase in mouse CD45 positive cells. In addition, SGR-2921 demonstrated dose-dependent inhibition of phosphorylated MCM2 in the spinal cord and spleen of the AML PDX models as surrogates of the bone marrow and extravascular compartments, respectively. Conclusions: Our data show remarkable dose-dependent in vivo activity of SGR-2921 in AML PDX models, including in those representing difficult-to-treat disease. Direct inhibition of CDC7 by SGR-2921 in AML blasts was demonstrated by a dose-dependent reduction of phosphorylated MCM2. Together, these data demonstrate that SGR-2921-mediated CDC7 inhibition is an attractive novel treatment opportunity in AML, with potential utility in patients with high risk mutations and relapsed and refractory AML.

Ex Vivo Anti-Leukemic Effect of Exosome-like Grapefruit-Derived Nanovesicles from Organic Farming-The Potential Role of Ascorbic Acid.

Citrus fruits are a natural source of ascorbic acid, and exosome-like nanovesicles obtained from these fruits contain measurable levels of ascorbic acid. We tested the ability of grapefruit-derived extracellular vesicles (EVs) to inhibit the growth of human leukemic cells and leukemic patient-derived bone marrow blasts. Transmission electron microscopy and nanoparticle tracking analysis (NTA) showed that the obtained EVs were homogeneous exosomes, defined as exosome-like plant-derived nanovesicles (ELPDNVs). The analysis of their content has shown measurable amounts of several molecules with potent antioxidant activity. ELPDNVs showed a time-dependent antiproliferative effect in both U937 and K562 leukemic cell lines, comparable with the effect of high-dosage ascorbic acid (2 mM). This result was confirmed by a clear decrease in the number of AML blasts induced by ELPDNVs, which did not affect the number of normal cells. ELPDNVs increased the ROS levels in both AML blast cells and U937 without affecting ROS storage in normal cells, and this effect was comparable to ascorbic acid (2 mM). With our study, we propose ELPDNVs from grapefruits as a combination/supporting therapy for human leukemias with the aim to improve the effectiveness of the current therapies.

Abstract 2122: The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML

Abstract The BRG1/BRM-associated factors (BAF) complex (also known as mSWI/SNF) is a critical regulator of the chromatin landscape of the genome. By controlling chromatin accessibility, BAF regulates lineage-specific transcriptional programs, including those important for AML blast cell growth and survival. FHD-286 is a highly potent inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4), which demonstrates strong tumor growth inhibition in several AML CDX and PDX models. FHD-286 is being developed for the treatment of relapsed/refractory AML and MDS (see Foghorn website for current status).Investigation of FHD-286 in a broad panel of AML cell lines showed that BAF inhibition affects hematopoietic transcriptional programs important for blast cell self-renewal and identity, agnostic of mutational background. Similarly, FHD-286 demonstrated broad efficacy in ex vivo treatment of AML patient-derived samples from diverse genetic backgrounds, including those with difficult to treat mutational profiles, including mtNPM1, FLT3 ITD, and Inv(3) with EVI1 overexpression. Interestingly, while higher concentrations (≥90 nM) of FHD-286 predominantly induced cytoreduction, lower concentrations (≤30 nM) predominantly led to differentiation-like responses. To investigate this differentiation effect, we performed immunophenotyping of cell lines and primary AML samples following prolonged treatment with FHD-286. Extended treatment (7+ days) with relevant concentrations (5-20 nM) of FHD-286 led to time- and dose-dependent upregulation of the myeloid differentiation marker CD11b, and acquisition of monocyte/metamyelocyte morphology. CD11b+ cells expressed lower levels of the hematopoietic transcription factor PU.1, as well as proliferation and survival proteins Ki67, Myc and BCL2. Additionally, primary patient samples showed a loss of CD34 positivity in blasts and GMP-like cells after 7 days of ex vivo treatment, indicating a decrease in stemness in these populations. Finally, upregulation of CD11b coincided with decreased BRG1 protein, suggesting that immature blasts are characterized by high levels of BRG1. These results suggest that BAF functions to drive transcriptional programs required to maintain AML cells in an undifferentiated state, and that FHD-286 may inhibit AML cell growth by overcoming this differentiation block. Expanding on these findings, we have also demonstrated synergistic activity with multiple combination partners, including cytarabine and decitabine, in vitro. Elaboration of this in both CDX and PDX in vivo models also shows significant survival benefit in difficult to treat mutational backgrounds. Taken together, these findings suggest that FHD-286 is able to target blast progenitor populations that are heavily BRM/BRG1-dependent, and that combination with standard of care agents can achieve profound, mutationally agnostic antitumor activity in AML. Citation Format: Mike Collins, Astrid Thomsen, Ashley Gartin, Gabriel J. Sandoval, Ammar Adam, Sarah Reilly, Laure Delestre, Virginie Penard-Lacronique, Warren Fiskus, Kapil Bhalla, Stephane de Botton, Sam Agresta, Jessica Piel, Murphy Hentemann. The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2122.

Electron Transport Chain Complex V Inhibition Enhances the Antileukemic Activity of Low-Dose Venetoclax in Acute Myeloid Leukemia

Background: BCL-2 dependent leukemia-stem cells preferentially rely on oxidative phosphorylation (OXPHOS) as a source of energy (Pei et al Cancer Discovery 2020; Stevens et al Nature Cancer 2021). BCL-2 inhibition through the use of venetoclax (VEN) has been linked to disruption of OXPHOS via reduced complex II activity. Given the use of OXPHOS by BCL-2 dependent AML cells, we sought to potentiate VEN effects by further perturbing electron transport in AML. Recently, we discovered that Ammocidin, a glycomacrolide natural product, targets leukemia OXPHOS via inhibition of ATP synthase, complex V of the electron transport chain (Reisman et al Nature Chem Bio 2022). Here, we explored the therapeutic window of simultaneous inhibition of Complex V and BCL-2 by ammocidin and VEN, respectively, in AML cells. Methods: We treated AML cell lines MV-4-11 and MOLM-13 with ammocidin and evaluated for viability via Cell Titer-Glo. To validate the combination of ammocidin and VEN, in vivo, we transplanted the MV-4-11 cell line into immunocompromised NSGS mice. Engrafted mice were treated with either standard of care (SOC) VEN (25 mg/kg 5x/week) + azacitidine (AZA) (0.2mg/ml 3x/week), ammocidin (0.1 mg/kg 5x/week) monotherapy, or a combination of each at these doses. Total human CD45+/CD33+ cells were removed from marrow, spleen and peripheral blood of xenografted mice and measured via flow cytometry. To further discover the efficacy of ammocidin/VEN in heterogenous AML samples, we treated primary patient AML cells with VEN, ammocidin or combination therapy. Mononuclear bone marrow cells from nine mutationally diverse patients with AML were tested and evaluated for cell viability via Cell Titer-Glo. To verify induction of apoptosis, we subjected cultured patient samples to annexin V/PI staining 24 hours after exposure to 300nM ammocidin and 100nM VEN single and combination therapy. Results: Cell Titer-Glo assays revealed that AML cell lines MV-4-11 and MOLM-13 cells were sensitive to complex V inhibition with a GI50 of 45 and 131nM. Combining ammocidin with VEN was synergistic in both cell lines. In vivo, the addition of ammocidin to an SOC regimen revealed significant decreases of AML cells in the bone marrow (SOC vs/ SOC+ ammocidin p=0.004), blood (SOC vs/ SOC+ ammocidin p=0.04) and spleen (SOC vs/ SOC+ ammocidin p=0.006). When looking at primary AML samples, combination therapy was more effective than single-agent therapy (Ven vs VEN+ ammocidin p=0.02). Induction of apoptosis, noted through Annexin+/PI− cell staining, was significantly enhanced with the combination therapy in AML blast cells at 24 hours (VEN vs VEN+ ammocidin, p=0.007). Further studies are underway to test this combination in both VEN sensitive and resistant AML PDX primagraft models in vivo. Conclusion: Targeting complex V and BCL-2 together resulted in the decreased expansion of human AML cell lines, and primary patient samples; and there was a preferential reduction of AML in vivo. These preliminary data represent a potential for ammocidin/VEN therapy in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Galectin 3 Protein Network Analysis By Reverse Protein Arrays Demonstrates Regulation of MCL-1, PKC Delta and CD74 Associated with Poor Survival in Patients with AML

Galectin 3 (LGALS3) is a glycan binding protein that positively regulates survival signaling by diverse mechanisms. LGALS3 is involved in trafficking of kinases such as RAS and receptors such as CD44 to membranes. LGALS3 gene expression is associated with poor survival in AML (Cheng et al Blood 2013) but its functional role is not clear. We therefore analyzed protein expression of LGALS3 and 230 other proteins by RPPA in 511 AML patients. LGALS3 was significantly elevated in AML blast cells compared to normal CD34+ counterparts and highest in monocytic AML patients. LGALS3 levels were highest in Intermediate and Unfavorable Cytogenetic groups. Pearson correlation of LGALS3 with the other proteins identified a distinct set of 23 proteins positively and 13 proteins negatively correlated with LGALS3 expression levels. LGALS3 positively correlated with the expression of anti-apoptotic BCL2 family member MCL-1. To validate RPPA results, OCI-AML3 cells were transduced with a lentivirus expressing shRNA against LGALS3 or with a control lentivirus expressing shRNA against a non-human gene (i.e. GFP). Suppression of LGALS3 in the cells results in reduced MCL-1 gene and protein expression. As reduction of LGALS3 by shRNA or inhibition of LGALS3 using GCS-100 suppresses phosphorylation of ERK, the mechanism how LGALS3 supports MCL-1 in the AML cells may involve ERK. OCI-AML3 LGALS3 shRNA cells were more sensitive to ABT-737 compared to control cells. RPPA also revealed that LGALS3 was strongly correlated with phosphorylated PKC delta and suppression of LGALS3 by shRNA reduced PKC delta phosphorylation levels implicating LGALS3 as a positive regulator of the kinase. While PKC delta is viewed foremost as a stress kinase, RPPA analysis of phosphorylated (i.e. active) PKC delta in AML patients reveals that elevated levels of active kinase is a prognostic factor associated with poor survival. The pro-survival role of PKC delta in AML may reflect its function as an AKT kinase or as a suppressor of ROS. RPPA also identified a novel positive association of LGALS3 with CD74. OCI-AML3 cells with LGALS3 shRNA did not exhibit altered CD74 expression suggesting that CD74 is upstream of the galectin or possibly that an as yet unidentified protein serves as a common regulator of both LGALS3 and CD74. Stromal cell derived CD74 has been implicated as important in the AML microenvironment as a regulator of IL-8 production (Abdul-Aziz et al Cancer Research 2016) but the role for CD74 in AML blast cells is not clear. CD74 modulates CD44 function so the protein could participate in signaling cascades involving the receptor. Pearson correlation with the other proteins analyzed identified 23 proteins including CD44 and LGALS3 that were positively correlated with CD74 and 8 proteins that were negatively associated with CD74. Though LGALS3 was not positively correlated with CD44, the galectin could potentially modulate CD44 function by influencing its surface expression as LGALS3 is involved in trafficking CD44 to the cell surface. The protein associations of LGALS3 and CD74 were used to assemble protein clusters that were then used for analysis of patient survival. LGALS3 and CD74 were collectively analyzed as part of a group of T-cell regulating proteins analyzed on this array. Within that group 4 different expression patterns (clusters) were defined by hierarchical clustering with LGALS3 being active in 2 of them. The protein cluster identifying active LGALS3 was prognostic for shorter remission duration. AML patients who exhibited both LGALS3 and CD74 active clusters exhibited very poor survival characteristics. For AML patients with both LGALS3 and CD74 active protein cluster, median overall survival was only 24.3 weeks, median remission duration was 17.8 weeks, and no patient survived beyond one year. These data suggest that LGALS3 supports AML cell survival via diverse pathways including positive regulation of MCL-1 and PKC delta and support of a CD74 protein network that identifies a new high risk AML population. It is our hope that strategies targeting LGALS3 and CD74 can be developed to help this new at risk population. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Drug Profiling Reveals Maturation Stage-Dependent Drug Responses in AML

IntroductionAcute myeloid leukemia (AML) is a disease of myeloid lineage precursors with poor overall survival and limited treatment options in relapsed patients. Ex vivo drug testing is an approach to identify potential responders for novel targeted agents. However, AML bone marrow is comprised of a mixture of malignant and non-malignant cell types, and may contain myeloid cells at distinct differentiation stages. Here, we evaluated the ex vivo sensitivity of primary AML samples at a detailed cell-population level using a multicolor high-throughput flow cytometer (FACS).MethodsBone marrow (BM) mononuclear cells of 23 AML patients were plated on 96-well plates in mononuclear cell media (PromoCell). Seven FDA approved drugs were used, including signal transduction inhibitors that had shown AML specific responses in our earlier studies (Pemovska et al. Cancer Discovery 2013 ): trametinib, ruxolitinib, everolimus, sunitinib, venetoclax, plus standard of care drugs idarubicin and cytarabine. After 3-day incubation, cells were stained with six cell surface markers (CD45, CD34, CD33, CD14, CD123, CD38) to identify major cell populations present in many AML samples: blasts, promonocytes/monocytes, lymphocytes and granulopoietic cells. Flow cytometric analysis was performed using the iQue Screener PLUS instrument (Intellicyt). Apoptotic and dead cells were removed from the analysis (Annexin-V/7-AAD) and live cell counts of different cell populations after drug treatment were measured. For comparison, the viability of the samples was also assessed using the CellTiter-Glo (CTG) assay that uses the amount of ATP as an indicator of metabolically active cells.ResultsImmunophenotyping of the AML samples showed large inter-sample variation in cell composition. The BM blast cell population out of the CD45+ positive cells ranged between 17-94% and the non-malignant lymphocyte population ranged from 0.4 to 46%. High numbers of promonocytes/monocytes were observed in samples from FAB M4/5 patients as expected, while FAB M0/1/2 patient samples mainly consisted of blasts and lymphocytes. FACS analysis of cell population specific drug responses revealed that blast cells of different patients had highly different drug sensitivities whereas lymphocytes and granulocytes did not show differences in drug sensitivities between the AML samples (Figure 1A). Interestingly, promonocytes/monocytes that are mainly present in FAB M4/5 samples but also in small quantities in some FAB M0/1/2 samples were systematically more sensitive to MEK inhibition, but more resistant to BCL-2 inhibition when compared to blasts (Figure 1A-B). In line with this observation, the average overall BM cell sensitivity of myelomonocytic/monocytic (FAB M4/5) samples as measured by CTG showed decreased sensitivity to venetoclax (P<0.01) and increased sensitivity to trametinib (P<0.05) when compared to less mature (FAB M0/1/2) AMLs. Initial differential gene expression analysis revealed enrichment of genes in the RAS/MAPK pathway in samples diagnosed with FAB M4/5. Amongst the tested compounds, venetoclax showed the highest toxic effect against blast cells even though 40% of the samples were at least partly resistant. Intriguingly, we observed that by combining venetoclax (50nM) with trametinib (25nM) or ruxolitinib (300nM), chemo- and venetoclax-resistant blast cells were effectively killed ex vivo (>80%) in all tested samples.ConclusionFACS-based drug testing ex vivo enables accurate drug sensitivity profiling of AML blast cells even in samples with high numbers of more mature leukemic cells and healthy cells. Our data indicate heterogeneity in drug responses of AML cells at different stages of myeloid differentiation, with high sensitivity to BCL-2 inhibition and resistance to MEK inhibition of the primitive blasts compared to more mature monocytic cells. MEK inhibitor sensitivity in FAB M4/5 AMLs might be linked to the inherent characteristics of monocyte lineage cells. Furthermore, we show that venetoclax-resistant blast cells can be effectively targeted by combining venetoclax with either MEK inhibitor trametinib or JAK inhibitor ruxolitinib. [Display omitted] DisclosuresMustjoki:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Celgene: Honoraria; Ariad: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Heckman:Novartis: Research Funding; IMI2 project HARMONY: Research Funding; Pfizer: Research Funding; Orion Pharma: Research Funding; Celgene: Research Funding.

Development of Foam-Based 3-Dimensional Culture to Investigate Drug Sensitivity in Primary Leukaemia Cells

IntroductionDevelopment of assays for evaluating leukaemia cells' sensitivity to antileukaemia agents is emerging to be an important aspect of personalized medicine. The conventional cell cultures lack the three-dimensional (3D) structure of the bone marrow (BM), the extracellular matrix and stromal components which are crucial for the growth and survival of leukaemia stem cells. To accurately predict the sensitivity of the leukaemia cells in an in vitro assay a culturing system containing the essential components of bone marrow is required. Various 3D cultures have been developed so far for studying leukaemia cells (Aljitawi et al . 2014). We hypothesize that a scaffold containing the spongious structure of the BM by providing the niche like spaces recapitulates the resistance to targeted therapy observed in a subset of leukaemia cells in vivo .Materials & MethodsA V-junction microfluidic device was used for bubble preparation. Calcium alginate foam-based scaffold were produced using sodium alginate solution (Ahmad et al. 2011) followed by adding Hydroxyapatite. Alginate microbubbles were collected in calcium chloride to cross-link the bubbles' shell. K562, AR230 and HL60 cell lines were cultured in RPMI medium supplemented with FBS, L-glutamine and penicillin/streptomycin. Cell proliferation and viability was measured by MTS assay. Primary normal CD34+ and acute myeloid leukaemia cells were cultured in StemSpan medium supplemented with CC100. Immunophenotype was determined using 8-color Euroflow AML/MDS antibody panel (CD16 FITC, CD13 PE, CD34 PerCP-Cy5.5, CD117 PE-Cy7, CD11b APC, CD10 APC-H7, HLA-DR V450, CD45 V500) for investigation of myeloid differentiation in normal CD34+ cells and myeloblasts from two AML patients. Data was acquired in a FACSCanto II flow cytometer (BD) and analysed with Infinicyt software (Cytognos).Results and conclusionK562 and HL60 leukemia cell lines were cultured in the porous alginate foam-based scaffold (Scanning Electron Microscopy image, top left figure) followed by evaluating their sensitivity to antileukaemia agents through measuring cell proliferation. Similar to BM niches, micro-cavities in the scaffold accommodated leukaemia cells (top right figure). Foam-based scaffold reduced the sensitivity of K562 and HL60 cells to inhibitory effect of imatinib and doxorubicin respectively compared to 2D culture (low left and right figures). Combination of foam-based scaffold with BM-derived stromal HS-5 cell line further reduced the leukaemia cells' sensitivity to antileukaemia agents. Foam-based 3D culture increased the differentiation of the haematopoietic cells compared to 2D culture as normal CD34+ precursors in the scaffold showed stronger and more homogenous expression of the myeloid associated antigens CD117 and CD13. Similarly, myeloblasts from AML patients in foam-based 3D culture showed a profound monocytic differentiation compared to 2D culture after 3 days. Similar to cell lines, primary CML CD34+ and primary AML blast cells showed increased resistance to imatinib and doxorubicin respectively in foam-based 3D compared to 2D culture (Figure). The conventional 2D culture is not a highly reliable system for drug sensitivity investigations as the in vitro drug sensitivity of leukaemia cells in 2D culture is different from what is observed in vivo (Edmondson et al . 2014). Our foam-based 3D culture reduced the leukaemia cells' sensitivity to antileukaemia agents and further induced the differentiation of the normal and leukaemia cells compared to 2D culture. These differences might be attributed to microcavities mimicking bone marrow niches in foam-based scaffold. We propose to apply this foam-based 3D culture for drug sensitivity investigations and therapeutic target discoveries. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single Cell Transcriptomics Revealed AML and Non-AML Cell Clusters Relevant to Relapse and Remission in Pediatric AML

Introduction: While advances in front-line conventional chemotherapy have increased the likelihood of attaining remission in pediatric AML, relapse rates remain high (25-35%), and novel therapies are needed (Zhang, Savage et al. 2019). The clinical and molecular heterogeneity of AML makes it complex to study and creates challenges for the development of novel therapies (Bolouri, Farrar et al. 2018). It is important to identify cells and pathways underlying relapse to facilitate development of novel therapies. Single-cell RNA Sequencing (scRNA-Seq) allows in-depth analysis of the heterogeneous AML landscape to provide a detailed view of the tumor microenvironment, revealing populations of blasts and immune cells which may be relevant to relapse or complete remission. Methods: We analyzed ~36,500 cells from 14 pediatric AML bone marrow samples in our institutional biorepository, spanning different AML subtypes and 3 healthy children to generate a comprehensive scRNA-Seq landscape of immature AML-associated blasts and microenvironment cells. Samples collected at the time of diagnosis (Dx), end of induction (EOI), and relapse (Rel) were used to generate scRNA-Seq data using a droplet-based barcoding technique (Panigrahy, Gartung et al. 2019). After normalization of scRNA-Seq data, the cell clusters were identified using principal component analysis and Uniform Manifold Approximation and Projection (UMAP) approach (Becht et al, 2018). Differential expression, pathways and systems biology analysis between relapsed and remission patients reveal differences for specific cell clusters (Panigrahy, Gartung et al. 2019). To determine the clinical outcome association of our AML blast specific markers, survival analysis was performed on AML TARGET data (https://ocg.cancer.gov/programs/target) using cox proportional hazard survival approach. To characterize AML blast cells with high accuracy, we used support vector machine (SVM), an Artificial Intelligence based feature extraction and model development approach (Bhasin, Ndebele et al. 2016). Results:ScRNA-Seq analysis of paired Dx and EOI samples using UMAP identified three blast cell clusters with significant gene expression differences among different patients, indicating heterogeneity of AML blast cells (Fig 1a, b). Comparative analysis of the three Dx enriched blast cell clusters with other cells identified a "core blast cell signature" with overexpression of genes like AZU1, CLEC11A, FLT3, and NREP (Fig 1c). These core AML-blast genes were linked to significant activation of the Wnt/Ca2+, Phospholipase C, and integrin signaling pathways (Z score &amp;gt;2 and P-value &amp;lt;.001). A subset of AML blast-specific genes also depicted significant association with poorer overall survival (CLEC11A Hazard ratio (HR)=1.9 ;95% CI=1.1-3.4;log-rank P=.03, FLT3 HR=2.4(1.5-3.9);P&amp;lt;0.001,NREP HR=1.9(1.2-3.1); P&amp;lt;0.008) (Fig 1d). Furthermore, we developed a highly sensitive 7-gene AML blast cell signature that distinguished AML blasts from normal myeloblasts and other hematopoietic cells (Area Under Curve of 0.94) using SVM. The scRNA-Seq of AML specific blast cells from relapsed and remission samples exhibited a different clustering pattern indicating different transcriptome landscapes. Relapse-associated AML cell clusters expressed high levels of AZU1, S100A4, LGALS1, and GRK2 genes (Fig 2a). Analysis of the non-AML tumor microenvironment demonstrated enrichment of T/NK in relapsed samples, with differential expression of T cell regulatory/activation genes (Fig 2b, c). ScRNA-Seq showed enrichment of monocyte/macrophage cell clusters in remission samples with distinct relapse- and remission-specific clusters. Remission associated macrophage/monocyte clusters showed overexpression of S100A10, FTH1, CST3 and IFITM2 genes (Fig 2d). Similarly, enrichment of T cell and monocyte/macrophage clusters was observed in relapse and remission samples respectively during EOI. Conclusions: Using single cell transcriptomics we developed a novel potential gene signature to characterize heterogenous AML blast populations with high sensitivity. These genes and the pathways they regulate implicate potential therapeutic targets in pediatric AML. Single cell transcriptome analysis also enabled identification of cell clusters with modulated gene expression at both Dx and EOI that may be useful in predicting relapse/remission. Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.

Acute myeloid leukemia–induced T-cell suppression can be reversed by inhibition of the MAPK pathway

AbstractAcute myeloid leukemia (AML) remains difficult to treat due to mutational heterogeneity and the development of resistance to therapy. Targeted agents, such as MEK inhibitors, may be incorporated into treatment; however, the impact of MEK inhibitors on the immune microenvironment in AML is not well understood. A greater understanding of the implications of MEK inhibition on immune responses may lead to a greater understanding of immune evasion and more rational combinations with immunotherapies. This study describes the impact of trametinib on both T cells and AML blast cells by using an immunosuppressive mouse model of AML and primary patient samples. We also used a large AML database of functional drug screens to understand characteristics of trametinib-sensitive samples. In the mouse model, trametinib increased T-cell viability and restored T-cell proliferation. Importantly, we report greater proliferation in the CD8+CD44+ effector subpopulation and impaired activation of CD8+CD62L+ naive cells. Transcriptome analysis revealed that trametinib-sensitive samples have an inflammatory gene expression profile, and we also observed increased programmed cell death ligand 1 (PD-L1) expression on trametinib-sensitive samples. Finally, we found that trametinib consistently reduced PD-L1 and PD-L2 expression in a dose-dependent manner on the myeloid population. Altogether, our data present greater insight into the impact of trametinib on the immune microenvironment and characteristics of trametinib-sensitive patient samples.

Abstract 3612: Decreased vitamin C uptake mediated by SLC2A3 promotes leukemia progression and impedes Tet2 restoration

Abstract Background: Two new studies in Nature and Cell, from Agathocleous et al. (2017) and Cimmino et al. (2017), respectively, showed that vitamin C regulates HSC function and suppresses leukemogenesis by modulating Tet2 activity. However, little is known about the relationship between vitamin C transporter and hematological malignancies. Purpose: In this study, we investigated the role of SLC2A3, a gene that encode GLUT3 which mainly transport glucose and oxidized form vitamin C, in hematological malignancies. Through this research we identified a set of biomarkers which can predict the effect of vitamin C treatment and develop stagey to improve this putative innovative treatment. Results: We analyzed gene expression patterns of the major GLUT family genes in primary AML blast cells using previously published microarray datasets (GSE37307) and (GSE9476) and determined that SLC2A3 gene expression was significantly decreased in blast cells compared with normal hematopoietic cells. By contrast, expression of SLC2A1, SLC2A2 and SLC23A2 was not significant different in between primary AML cells and normal hematopoietic cells. Next, we analyzed the relevance of SLC2A3 expression to the survival of AML patients from TCGA and TARGET database and found that below-median SLC2A3 expression was associated with inferior overall survival. Subsequently, we detected the SLC2A3 mRNA expression level on 4 AML cell lines and 3 DLBCL cell lines, there were very low expression level on OCI-AML-3 and OCI-LY1. To check whether decreased expression of SLC2A3 has an impact on the Vitamin C effect, we treated the 7 cell lines with dose dependent vitamin C, up to 500 μM, 48 hours. The cell viability assay showed that OCI-AML-3 and OCI-LY1 have no response to Vitamin C, however the proliferation of NB4, HEL, HL60, OCI-LY19 and Toledo was suppressed by 250 μM Vitamin C. Conclusions: SLC2A3 expression was decreased in AML blast cell and down regulation of SLC2A3 was closely associated with the poor outcomes of AML patients. In vitro study showed that SLC2A3 was essential for the effect of Vitamin C. Therefore, SLC2A3 could be a potential biomarker to predict the effect of Vitamin C treatment. In addition, through targeting SLC2A3, efficacy of Vitamin C treatment could be improved. Citation Format: Jun Liu, Junshik Hong, Kwangsung Ahn, Sungsoo Yoon. Decreased vitamin C uptake mediated by SLC2A3 promotes leukemia progression and impedes Tet2 restoration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3612.

Abstract 4828: Anti-tumor activity of BAY-943, an anti-IL3RA ADC with a novel KSP inhibitor payload, in CDX and PDX AML models

Abstract Despite recent progress in the treatment of AML, clinical outcomes have improved only minimally over the past three decades. Therefore, novel therapeutic agents with a high therapeutic window and a favorable tolerability profile are urgently needed to improve the therapeutic outcome for AML patients. IL3RA (CD123) is the alpha subunit of the interleukin 3 (IL-3) receptor which regulates proliferation, survival and differentiation of hematopoietic cells. IL3RA is expressed at high frequency, with ~84% of AML cases and 59% of classical Hodgkin lymphoma (cHL) cases being positive. IL3RA is expressed in AML blast and leukemic stem cells (LSCs) but not in hematopoietic stem cells (HSCs). In healthy individuals, the IL3RA expression is restricted to myeloid progenitor cells, plasmacytoid dendritic cells (pDCs), basophils and - at low levels - monocytes and B-lymphocyte subsets. This expression pattern suggests that IL3RA could be a clinically relevant target for an antibody-drug conjugate (ADC) approach in treatment of AML, cHL, and MDS. BAY-943 is a novel antibody-drug conjugate (ADC) consisting of a humanized internalizing anti-IL3RA IgG1 antibody (Ab, EC50 on IL3RA-positive tumor cells in flow cytometry: 2-5 nM) conjugated via lysine residues to a potent proprietary kinesin spindle protein inhibitor (KSPi). The kinesin spindle protein (KSP/Eg5/KIF11) is essential for the proper segregation of duplicated centrosomes during spindle formation in the G2/M phase of the cell cycle, as such it is only active in proliferating cells. In vitro, in a panel of IL3RA-positive AML and HL cell lines, BAY-943 showed potency in the nano- to subnanomolar range. In IL3RA-positive cell line derived (CDX) AML xenograft models (MOLM-13 and MV4-11) and patient-derived xenograft (PDX) models, BAY-943 dosed at 10 mg/kg given Q7Dx increased survival compared to vehicle treated mice. Tumor burden (percentage of human CD45 positive AML cells) was significantly reduced compared to vehicle treated mice. In the subcutaneous IL3RA-positive cHL CDX model HDLM-2, BAY-943 dosed at 5 and 10 mg/kg Q7Dx2 induced complete tumor remission in 12 out of 13 mice. In safety studies in Cynomolgus monkeys, BAY-943 (which is cross-reactive with Cynomolgus IL3RA), up to 20 mg/kg single or 10 mg/kg repeat (QWx3) dose were well tolerated with no signs of thrombocytopenia, neutropenia and no liver toxicity, i.e. adverse events observed with ADCs containing other payload classes. As expected, a transient reduction of IL3RA expressing cell types (basophils, pDCs) was observed. In summary, IL3RA-KSPi-ADC BAY-943 shows efficacy in IL3RA-positive AML and HL models and has a favorable safety profile in monkey repeat dose studies. Overall, the preclinical results support further development of BAY-943 as an innovative approach for the treatment of IL3RA-positive AML. Citation Format: Anette Sommer, Dennis Kirchhoff, Antje M. Wengner, Beatrix Stelte-Ludwig, Hans-Georg Lerchen, Anne-Sophie Rebstock, Oliver von Ahsen, Lisa Dietz, Pascale Buchmann, Sandra Johanssen, Dominik Mumberg, Bertolt Kreft. Anti-tumor activity of BAY-943, an anti-IL3RA ADC with a novel KSP inhibitor payload, in CDX and PDX AML models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4828.

PS988 CHARACTERIZATION OF A NOVEL, POTENT SMALL MOLECULE MDM2 ANTAGONIST WHICH ACTIVATES WILD‐TYPE P53 AND INDUCES APOPTOSIS IN AML

Background:In the presence of various stress signals, p53 acts as a tumor suppressor by regulating the expression of a multitude of genes to elicit cellular responses such as cell cycle arrest and apoptosis. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that acts as a primary inhibitor of p53 function by, for example, targeting p53 for proteasomal degradation. Early studies have demonstrated that blocking the MDM2‐p53 interaction in tumors carrying wild‐type p53 prevents p53 degradation and reactivates it. Small molecule MDM2 antagonists that inhibit the MDM2‐p53 interaction, therefore, present a promising strategy for cancer therapy and a number of these compounds are in clinical development.Aims:Herein, we describe the characterization of a novel, potent small molecule MDM2 antagonist in AML in vitro and in vivo pre‐clinical models and in patient‐derived AML blast cells.Methods:A panel of p53 wild‐type AML cell lines was tested for reduction in cell proliferation using Alamar Blue assay following treatment with the compound. Induction of apoptosis was measured by flow cytometry using a fluorescent caspase‐3 substrate or Annexin V. Target engagement was analyzed by Western Blotting and TaqMan qRT‐PCR. The MV‐4‐11 mouse systemic model was used to test in vivo sensitivity to the compound. Primary AML blasts were isolated from patients using combinations of antibodies against CD34, CD33, CD45, and CD117.Results:We have applied structure‐based design to develop a novel, potent, orally bioavailable MDM2 antagonist. The compound exhibits EC50 &lt;1 nM against the full‐length MDM2 protein in a cell‐free ELISA and increases p53 levels in a wide range of p53 wild‐type cells (e.g. EC50 = 10 nM for p53 induction in SJSA‐1 osteosarcoma cells).When tested in a panel of p53 wild‐type AML cell lines, the compound exerted a strong anti‐proliferative effect with GI50 values of &lt;30 nM being observed in 9 out of 11 cell lines. In contrast, the compound had little effect on p53 mutant KG‐1 cells (GI50 &gt;10 mM). In addition, many of the p53 wild‐type AML cell lines showed a strong induction of apoptosis in response to treatment with the compound. Activation of p53 was evident by an increase in the expression of p53 and that of its well‐known transcriptional targets such as p21 and MDM2. Consistent with these findings, a detailed study of gene expression changes in MV‐4‐11 confirmed clear transcriptional activation of several p53 target genes (CDKN1A, MDM2, BBC3, FAS, GADD45, BAX) 2–6 hours after addition of the compound.In accordance with its potent activity in vitro, the compound displayed significant in vivo efficacy in the MV‐4‐11 mouse systemic model of AML. Here, QDx14 oral dosing at well tolerated doses demonstrated a clear reduction in tumor burden. Furthermore, p53 activation by the compound triggered apoptosis when tested in primary AML blast cells isolated from patients.Summary/Conclusion:Taken together, our findings demonstrate that the compound exhibits potent activity against AML cells that retain wild‐type p53, thus meriting further clinical investigations.

Stemness Signature in AML: GEP with 17 Genes Score Versus Leukemic Stem Cell (LSC) Quantification By Multiparameter Flow Cytometry (MFC)

Background: Clonal heterogeneity of the AML blast cell population has been well documented using next generation sequencing (NGS) strategies. This heterogeneity extends to the presence of LSC fraction among the leukemic population: a dynamic compartment evolving to overcome cell selection pressure during disease progression and its treatment including allogeneic Hematopoietic Stem Cell Transplantation (HSCT). However, it remains difficult to discriminate between LSC and nHSC or other myeloid progenitors as MPP, LMPP or GMP. Here we present how to combine FLOWSom and KALUZA software to obtain robust identification among tumoral and normal progenitors. We compared the flowLSC Profile with stemness 17 genes score to identify patients with adverse outcome.Methods: 11 AML patients from ALFA07-01 clinical trial diagnosed in Lyon University Hospital between 2009-2010 were included in this study. The quantification of CD34+CD38- fraction at diagnosis from total bulk leukemia was made as previously described. To better discriminate between LSC and nHSC we designed MFC panel based on 2-tube antibody combinations to identify the pattern of LSC in the CD34+CD38- cell compartment. CD34/CD38/CD45/CD117« backbone » was used in both tubes, completed by lineage defined markers CD7/CD56/CD19/CD13/CD33/ and/or LSC identified markers CD123 +/- CD90/CD45RA, CLL1, TIM3, CD97.Results: As seeing in Table Nr 1, between 11 AML patients we observed a strong correlation identifying patients with adverse prognostic using previously published threshold by Flow (> 1% CD34+CD38- from bulk leukemia cells). 4 from 11 patients showed a discrepancy between GES 17 LSC and Flow CD34+CD38-: one from four patients (Nr 3) was classified as “high” by Flow (high CD34+ expression) and “low” by GES LSC 17 and three other patients (Nr 8, 10, 11) were classified inversely as “low” by Flow (<1% CD34+ expression) and “high” by GES LSC 17 scoring. Our preliminary data tend to show the relevance to establish a scoring system including the level of FlowLSC at diagnosis and GES LSC 17 to distinguish patients with high risk in patients with high CD34+ expression.Conclusion: In summary, we confirmed that the presence of the LSC CD34+CD38- subpopulation representing more than 1% of total “bulk leukemic cells” at diagnosis could help to identify patients with a poor outcome. Using more accurate analysis as FLOWsom “neural network” system we showed the better discrimination between nHSC and LSC pathway with better separation between CD34+CD38- and CD34+CD38lo fractions and between nHSC (Fig 1A) and LSC (Fig 1B). This unsupervised system could allow us to improve and harmonize the analysis of AML LSC flow signature at diagnosis. A high LSC17 score probably reflects biological properties of LSCs that confer resistance to standard AML therapy. The previous analysis of the ALFA- 0701 trial data demonstrates the utility of the LSC17 score as a tool for patient selection. Despite heterogeneity and complexity of the AML LSC compartment, we should still use LSC quantification as a biomarker of chemosensitivity of AML (Fig 1C). For the patients with high-level of LSC other therapeutic modalities should be chosen to eradicate LSC using targeting immunotherapy before allograft. Monitoring of the LSC fraction should be useful in most clinical trials to overcome chemoresistance of LSC. These results should be confirmed in a prospectively larger cohort of patients and could be considered as useful complementary prognostic parameter for risk-stratification AML patients in future clinical trials.Image: Fig1 A, B, C Table 1Clinical Trial Registry: ALFA 0701Conflict of Interest: No conflictKey-Words: LSC, flow cytometry, AML, LSC17 Scoring

A phase 1, open-label, dose-escalation, multicenter study to evaluate the tolerability, safety, pharmacokinetics, and activity of ADCT-301 in patients with relapsed or refractory CD25-positive acute myeloid leukemia.

TPS7071Background: In normal human tissue, CD25 expression is mainly limited to activated T- and B-cells. Expression of CD25 by AML blast cells is associated with adverse outcomes, including induct...

Nuclear Export Inhibitor KPT-8602 Is Highly Active Against Leukemic Blasts and Leukemia-Initiating Cells in Patient-Derived Xenograft Models of AML

Currently available combination chemotherapy for AML induces complete remission in about 75% of younger and 45% of older patients, but often fails to induce long-term disease-free survival and is toxic to normal hematopoietic cells. The frequent disease relapse observed in chemotherapy treated AML patients is thought to occur through the inability of the existing drugs to specifically target the self-renewing leukemia-initiating cells (LICs). These cells have the functional capacity to replenish AML blasts. An attractive target for AML therapy is the nuclear export protein exportin 1 (XPO1), which is functionally required for continuous nuclear export of a subset of proteins and mRNAs. Small molecule Selective Inhibitor of Nuclear Export (SINE) compounds covalently bind to Cysteine528 in the cargo-binding groove of XPO1 and inhibit nuclear export. The orally bioavailable clinical SINE compound, selinexor (KPT-330), is currently in Phase 1 and 2 clinical trials in adult patients with AML (NCT01607892, NCT02088541, NCT02249091, NCT02403310, NCT02093403, NCT02485535, NCT02299518, NCT02416908, NCT02212561, and NCT01607892) and in a Phase 1 trial for relapsed childhood ALL and AML (NCT02091245). The results of these trials are encouraging, as they have demonstrated that selinexor alone or in combination is active in inducing remission in patients with relapsed or refractory AML. Here, we explore the anti-leukemia activity of a next generation SINE compound, KPT-8602.In preclinical toxicology studies as well as clinical trials, noted adverse events for selinexor are anorexia with weight loss, fatigue and thrombocytopenia, which limit the frequency of dosing to no more than every other day, three times a week. KPT-8602 is orally bioavailable and has similar pharmacokinetic properties to selinexor, with reduced brain penetration. Preliminary toxicology studies in rats and monkeys suggest that KPT-8602 has a substantially better tolerability profile, with reduced fatigue, thrombocytopenia, and anorexia compared to selinexor. Thus, KPT-8602 can be dosed orally on a daily basis for 5 days each week. The increased drug exposure with daily dosing and better tolerability of KPT-8602 leads directly to superior efficacy in pre-clinical models compared with selinexor.To define the anti-leukemia activity of KPT-8602 against primary AML blasts and LICs in a relevant pre-clinical setting, we established patient-derived xenograft (PDX) models, in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcgnull (NSG) mice. Mice engrafted with leukemic blasts were treated with vehicle, selinexor (20 mg/kg three times per week), or KPT-8602 (15 mg/kg daily for 4 weeks). KPT-8602 was highly active against blast cells from three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN), AML with complex karyotype (AML-CK), and MDS-derived AML (MDS/AML)), as evidenced by a reduction in leukemic engraftment in primary mice after treatment (Fig. 1A-C). At these doses, KPT-8602 demonstrated higher anti-leukemic activity than selinexor, with no leukemic cells detected in the bone marrow of two of the eight AML-CN PDX treated mice (Fig. 1A-C). In secondary transplantation assays, KPT-8602 greatly reduced the frequency of LICs in the PDX model derived from AML-CN cells, indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs (Fig. 1D). In mice engrafted with normal cord blood human CD34+ cells, KPT-8602 reduced bone marrow human CD45+ cell numbers (Fig. 1E), but the impact on normal hematopoietic stem and progenitor cells, as determined by secondary transplantation assays (Fig. 1E), was modest when compared to the LIC reduction after KPT-8602 treatment. These findings demonstrate that KPT-8602 has better tolerability, perhaps through decreased CNS penetration, and is better at eliminating both AML LIC and blast cells when compared to selinexor. Therefore, KPT-8602 should be tested for efficacy and tolerability in patients with relapsed and refractory AML, with the goal to overcome a difficult obstacle in curing AML; destroying the LIC compartment while sparing normal hematopoietic cells. [Display omitted] DisclosuresEtchin:Karyopharm: Research Funding. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Ellis:Karyopharm Therapeutics Inc: Employment. McCauley:Karyopharm Therapeutics, Inc: Employment. Stone:Agios: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Karyopharm: Consultancy; Celator: Consultancy; Novartis: Research Funding; Merck: Consultancy; Juno: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Roche/Genetech: Consultancy; Celgene: Consultancy. DeAngelo:Bristol Myers Squibb: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Ariad: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership. Look:Karyopharm Therapeutics, Inc: Research Funding.

Low Vitamin D Levels Are Associated with Inferior Survival Following Azacitidine Treatment in Patients with Myelodysplastic Syndrome

▪Introduction: Azacitidine (AZA) therapy has become the recommended first-line treatment for patients with high-risk myelodysplastic syndromes (MDS) and oligoblastic acute myeloid leukemia (AML; bone marrow blasts <30%). However, only around 50% of patients achieve objective responses with AZA therapy and the vast majority of responding patients have disease progression within 2 years resulting in dismal survival rates.Vitamin D (VitD) is a central regulator of calcium and bone homeostasis and affects multiple signaling pathways controlling proliferation, apoptosis and differentiation. More than 30 years ago, VitD was demonstrated to induce in vitro differentiation of AML blast cells. Consequently, VitD alone or in combination with other differentiating agents has been used for treatment of MDS patients showing inconsistent and rather limited efficacy. We retrospectively tested the hypothesis that VitD levels prior to start of treatment are predictive of overall survival (OS) in newly diagnosed MDS and oligoblastic AML patients receiving upfront AZA therapy. In addition, the antiproliferative effects of AZA in combination with different VitD derivatives were investigated in vitro.Patients, materials and methods: A total of 58 patients treated at our center between 2006 and 2014 had serum samples available for assessment of VitD status. Serum samples were collected at a median of 18 days prior to start of AZA treatment and cryopreserved at -80°C. VitD status was assessed by measurement of circulating 25-hydroxyvitamin D levels applying a standard chemiluminescent immunoassay (DiaSorin Deutschland GmbH, Dietzenbach, Germany). Overall survival (OS) was estimated using the method of Kaplan and Meier. Survival times were measured from the date of AZA treatment start. Comparison of OS between the VitD groups was done using the logrank test and by cox regression adjusting for known confounders.For in vitro analyses, a tetrazolium based MTT assay was used for assessment of growth inhibition of two different AML cell lines (HL60 and MOLM13) after exposure to AZA alone or in combination with VitD (1alpha,25-dihydroxyvitamin D and 25-hydroxyvitamin D; both from Cayman Chemical, Ann Arbor, MI, USA) or the VitD antagonist TEI-9647 (kindly provided by Teijin Pharma Ltd., Tokyo, Japan). Drug interactions were analyzed using the median-effect method of Chou and Talalay and combination index (CI) values were calculated according to the classic isobologramm equation with CI<1, CI=1 or CI>1 corresponding to synergism, additivity or antagonism, respectively. In cases where no antiproliferative activity upon single-agent treatment was assessable (low-dose VitD and TEI-9647), an analysis in terms of sensitization (potentiation) or inhibition of AZA activity was performed instead.Results: Estimated median follow-up time was 14.2 months. Median serum VitD level prior to AZA treatment was 33 nM (range 11-102 nM). A total of 18 patients underwent allogeneic stem cell transplantation (alloSCT) following AZA therapy and follow-up was censored at the time of alloSCT. Patient, disease and treatment characteristics did not differ significantly between the low (≤33 nM; n=29) and high (>33 nM; n=29) VitD group. Estimated one-year survival was 72% (95% CI 54-90%) in the high VitD group, which was significantly longer as compared to the low VitD group (41%, 95% CI 20-62%, p<0.05; Figure 1). In multivariate analysis with OS as endpoint, VitD (per 10 nM decrease, HR 1.83 95% CI 1.06-3.16, p=0.03) and adverse cytogenetics (HR 2.58 95% CI 1.10-6.06, p=0.03) were independent predictors of shorter survival. In vitro treatment of HL60 and MOLM13 cells with AZA in combination with VitD produced synergistic and additive antiproliferative effects. Addition of nanomolar concentrations of VitD to AZA resulted in potentiation of AZA activity. Conversely, combination with TEI-9647 resulted in inhibition of AZA activity.Conclusions: Our study suggests that higher VitD levels were associated with a survival advantage following upfront AZA therapy. As compared to AZA monotherapy combination treatment with VitD resulted in enhanced cytotoxic effects in vitro. VitD repletion/supplementation during AZA treatment should therefore be explored. [Display omitted] DisclosuresLuft:Immundiagnostik AG: Research Funding.

Immunotherapy with Long-Lived Anti-CD123 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human AML Cell Lines and of CD123+ Cells in Monkeys: A Potential Therapy for Acute Myelogenous Leukemia

CD123 (IL-3 receptor alpha) is highly expressed on acute myeloid leukemia stem cells and blasts, and represents a promising target of antibody therapies for AML. Anti-CD123 antibodies such as CSL-362 and KHK2823 are currently in clinical development; however, a limitation of these molecules is that they are unable to stimulate T cell-mediated killing of CD123+ AML cells. To exploit the potent activity inherent to T cell immunotherapy while maintaining the favorable dosing regimen of a therapeutic antibody, we have designed a novel bispecific antibody that recruits T cells to attack CD123+ AML stem and blast cells. Such antibodies act via a mechanism known as “redirected T cell-cytotoxicity” (RTCC), because they stimulate targeted T cell-mediated killing regardless of T cell antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).We first generated a library of humanized and affinity-optimized anti-CD123 × anti-CD3 bispecific antibodies and assessed their potency using RTCC assays, in which bispecifics stimulated killing by human T cells of the CD123+ AML cell lines KG-1a and TF-1. From this cell-based screen, we selected the bispecific antibody XmAb14045 for testing in animal models. This antibody has 0.1 nM affinity for human CD123, and a T cell-engaging domain with 8 nM affinity for human CD3. XmAb14045 stimulated T cell-mediated killing of KG-1a and TF-1 cells with an EC50 < 1 ng/ml (8 pM). In contrast, XmAb14045 had no cytotoxic activity against the CD123− Raji B cell line, demonstrating target specificity of the T cells.XmAb14045 had a prolonged serum half-life in mice of 6.2 days, in marked contrast to non-Fc domain-containing bispecific formats. Because this antibody was optimized for human CD123 and CD3 targets and does not crossreact with mouse antigens, we evaluated efficacy in cynomolgus monkeys. We treated 3 monkeys per group with a single dose of XmAb14045 at 0.01, 0.1, or 1 mg/kg. We quantified CD123+ cell numbers, including basophils and plasmacytoid dendritic cells (pDC) as CD123+ surrogate populations for AML stem and blast cells. Within 4 hours of dosing, XmAb14045 strongly activated T cells and stimulated depletion of over 99% of circulating CD123+ cells within 1 hr, particularly at the 0.1 and 1 mg/kg doses. Basophil and pDC counts fell to baseline within 4 hr and remained low for several weeks. Circulating CD4+ and CD8+ T cells were activated immediately after dosing and this was sustained for 48 hr, as measured by markedly increased levels of the activation markers CD25 and CD69. Notably, XmAb14045 induced rapid margination of CD4+ and CD8+ T cells from the circulation, with blood T cell populations returning to baseline within several days. Bone marrow CD123+ cells were depleted by over 95% at all doses, and these cell populations had not recovered by 8 days after treatment.Our results demonstrate that bispecific antibodies can recruit and activate T cells to efficiently kill CD123+ cells not only in the circulation but also in the bone marrow. Results in monkeys also suggest that changes in basophil and/or plasmacytoid dendritic cell numbers are readily quantifiable in peripheral blood, and thus these populations may serve as biomarkers for clinical efficacy. Our preclinical data provide a rationale for clinical assessment of anti-CD123 × anti-CD3 bispecific antibodies in patients with acute myeloid leukemia. DisclosuresChu:Xencor: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

PKR Inhibits the DNA Damage Response and Correlates with Poor Clinical Outcomes in AML

Introduction:Dysregulation of the double-stranded RNA-activated protein kinase (PKR) disturbs cellular signaling pathways that influence proliferation, differentiation, inflammation and apoptosis. Given the multifaceted functions of PKR in cellular stress signaling and inflammation, how PKR may play a role in tumorigenesis is a significant but underexplored area. Using a proteomic approach, we stratified AML patients according to PKR expression level in CD34+ leukemic cells and find that patients with high PKR expression have inferior clinical outcomes. Significantly, results from both acute leukemia derived cell lines and primary AML BM samples reveal that increased PKR expression leads to inhibition of DNA damage response (DDR) and DNA double strand break (DSB) repair. Thus, high PKR expression/activity in leukemic cells may retard the DNA damage response, enhance genomic instability and facilitate leukemia progression.Method:Peripheral blood and bone marrow specimens were collected from 414 patients with newly diagnosed AML and evaluated for outcomes at The University of Texas M.D. Anderson Cancer Center (MDACC) between September 1999 and March 2007. Samples were acquired during routine diagnostic assessments in accordance with the regulations and protocols (Lab 01-473) approved by the Investigational Review Board (IRB) of MDACC and analyzed under IRB-approved laboratory protocol (Lab 05-0654). Transgenic PKR (TgPKR) and dominant-negative PKR (DNPKR) mice were previously developed in our lab. PKR knockout mice (PKRKO) were provided by Dr. Robert Silverman (Lerner Research Institute, Cleveland Clinic, Cleveland, OH). The study was approved by UF IACUC protocol #201102224. Sample preparation and RPPA processing was done as described (Kornblau et al., 2011). Proteomic profiling for PKR was performed on AML patient samples by RPPA using the PKR monoclonal M02 antibody (Abnova). Neutral Comet assay and Olive tail moment calculations were performed by Wimasis Image analysis (Munich, Germany). γ-H2AX, p-ATM, p-NBS-1 levels were evaluated by both western blot and flow cytometry.Results:High level of PKR expression in CD34+ blast cells from 414 newly diagnosed AML patients correlates with reduced remission duration and poor survival. Cells with decreased PKR expression or treated with the PKR inhibitor (PKRI) have a significantly more rapid induction of γ-H2AX formation, increased p-ATM and p-NBS1 following ionizing irradiation (IR). On the other hand, bone marrow hematopoietic stem progenitor cells (HSPCs) isolated from transgenic mice overexpressing human PKR (TgPKR) display a decreased level of p-ATm and p-NBS1 indicating a depressed DDR following IR. Significantly in human CD34+ leukemia blasts an inverse relationship exists between PKR expression and γ-H2AX or p-ATM. Results from the Neutral comet assay shows that REH cells with decreased PKR expression or HSPCs from PKRKO mice are able to repair DSBs at a significantly faster rate than HSPCs from WT mice, which repair DSB at faster rate than those from TgPKR mice. In addition treatment of TgPKR cells or CD34+ AML blasts with PKRI can restore the efficiency of DSB repair.Conclusions:Our findings demonstrate that PKR inhibits/retards the DDR and DNA DSB repair following genotoxic stress, the consequence of which results in genomic instability. This finding may explain, at least in part, our observation that high PKR expression in primary AML blast cells is associated with poor survival of patients with AML. Therefore pharmacological inhibition of PKR may represent a novel therapeutic strategy that targets and reduces genomic instability in these cells and may improve the clinical outcomes in such AML patients. DisclosuresNo relevant conflicts of interest to declare.

Abstract 662: Generation and characterization of antibodies specific for IL1RAP antigen to target quiescent and proliferating AML leukemic stem cells

Abstract We have isolated and characterized blast cells and leukemic stem cells (LSCs) enriched from AML patients and identified cell surface targets selectively expressed on AML blast cells and LSCs and not their normal hematopoietic stem cell counterparts. A series of potent, specific, monoclonal antibodies (mAbs) have been generated against one of these AML LSC targets, IL1RAP. IL1RAP mAb clones were characterized for patient binding properties and also evaluated in in vitro cell based assays and in in vivo tumor xenograft models. These monoclonal antibodies demonstrated promising binding profiles in primary AML patients’ samples and robust anti-tumor activity in in vitro cell based assays and in vivo tumor xenograft models. Specifically, select clones have a high degree of specific binding to AML primary patient samples (&amp;gt;90%), and specifically kill primary patient AML cells in complement-dependent cytotoxicity assays. These IL1RAP clones are being evaluated for inhibition of clonogenic potential of AML patient samples in vitro in colony forming assays and against normal clonogenic hematopoietic bone marrow derived stem cells. Select IL1RAP mAb clones evaluated in orthotopic AML tumor xenograft models have shown potent inhibition of tumor engraftment as well as significant tumor growth inhibition effects ranging from 80-90%. Because a key driver of LSC resistance is their quiescence, we also evaluated the antitumor effect of IL1RAP mAbs on quiescent AML cells in vitro. In summary, the patient binding profiles and in vitro and in vivo efficacy against AML cells indicates that targeting IL1RAP is a promising therapeutic approach for the treatment of AML and has the potential for effective eradication of AML LSCs and long term remission in the clinic. Citation Format: Ping Jiang, Sharmili Roy, Jen Huang, Trang Dao-Pick, Jennifer Lu, Madhavi Mishra, Jeffrey Lin, Eric D. Hsi, Robert J. Tressler, Holger Karsunky. Generation and characterization of antibodies specific for IL1RAP antigen to target quiescent and proliferating AML leukemic stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 662. doi:10.1158/1538-7445.AM2014-662