Normal Human Bone Marrow Cells Research Articles

Abstract 3333: Targeting the MDM2-p53 interaction: Time- and concentration-dependent studies in tumor and normal human bone marrow cells reveal strategies for an enhanced therapeutic index

Abstract Aim We aimed to design an MDM2-p53 antagonist with a differentiated tolerability profile that could be used to treat patients with wild-type TP53 malignancies. As part of an alliance between Newcastle University, Astex Pharmaceuticals, and Cancer Research Horizons, we discovered ASTX295, a potent inhibitor of the MDM2-p53 interaction that is currently under clinical investigation in patients with solid tumors (NCT03975387). We selected ASTX295 as a compound with a predicted short plasma half-life, which we hypothesised would help to mitigate the dose-limiting neutropenia and thrombocytopenia observed with earlier MDM2-p53 antagonists in clinical studies. To examine this hypothesis in vitro, we determined time- and concentration-dependent responses to ASTX295 treatment in healthy volunteer-derived human bone marrow cells, megakaryocytes, and in a panel of human tumor cell lines. Methods Samples containing bone marrow cells from healthy patients undergoing hip surgery were obtained under the ethical approval of the Newcastle Biobank (REC 12/NE/0395). Following Lymphoprep™ separation, cells were treated ex vivo with ASTX295 and seeded for Granulocyte-macrophage (GM) colony-forming assays in methylcellulose. Megakaryocytes were obtained from in vitro differentiation of CD34+ stem/progenitor cells. Human tumor cell lines (including MDM2-amplified SJSA-1) were treated with ASTX295 in vitro and seeded at low density for colony-forming assays. Exposures of 6, 12, or 24h were examined, and the data plotted to calculate LC50 values. Results The clonogenic survival of tumor cells was time-dependent, with LC50 values (mean ± SEM) in SJSA1 cells being 238 ± 46nM and 75 ± 7nM respectively (n = 3-4), following a 12h or 24h exposure to ASTX295. Time-dependent effects were also evident in five human bone marrow samples but with LC50 values of 1.9, >3, >10, >10, and >10uM being achieved at 12h, and 860 ± 268nM at 24h. Megakaryocytes showed similar time-dependent sensitivities in which daily treatment of 2 or 6h over three days did not induce apoptosis while significant cell death was observed when the treatment time was extended to 16-24h daily. In contrast, short, daily pulse treatment of 2-6h in cell lines (MV4-11, MOLM-13, SJSA-1) over three days was sufficient to induce cell death. Conclusions ASTX295 is a potent antagonist of the MDM2-p53 interaction. Collectively, our in vitro data suggest that a shorter exposure to ASTX295 (up to 12h), may help to spare healthy bone marrow cells whilst killing tumor cells. Hence, intermittent exposure to an MDM2-p53 antagonist could favourably modulate its therapeutic index. The predicted short plasma half-life of ASTX295 should provide flexibility in controlling the duration of exposure in vivo, potentially enabling a more bone-marrow sparing approach to MDM2-p53 antagonism to be utilised. Citation Format: Elaine Willmore, Maria Ahn, Suzanne Kyle, Yan Zhao, Huw Thomas, Kenneth S. Rankin, Luke Bevan, Lynsey Fazal, Keisha Hearn, Nicola Wilsher, Justyna Kucia-Tran, Nicola Ferrari, Nicola Wallis, Neil Thompson, John Lyons, Duncan Miller, Celine Cano, Martin E. Noble, Ian R. Hardcastle, Steven Howard, Gianni Chessari, John Lunec, David R. Newell, Steve R. Wedge. Targeting the MDM2-p53 interaction: Time- and concentration-dependent studies in tumor and normal human bone marrow cells reveal strategies for an enhanced therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3333.

RTID-05. SURGICAL WINDOW OF OPPORTUNITY TRIAL REVEALS MECHANISMS OF RESPONSE AND RESISTANCE TO NAVTEMADLIN (KRT-232) IN PATIENTS WITH RECURRENT GLIOBLASTOMA

Abstract Over half of glioblastoma multiforme (GBM) patients retain wild-type p53, resulting in potential susceptibility to MDM2 inhibitors. These molecules disrupt the interaction between p53 and its negative regulator MDM2, resulting in downstream pathway signaling and cell fate decisions such as growth inhibition and death from cellular stress. We have analyzed clinical samples from 21 patients with recurrent GBM treated with the MDM2 inhibitor KRT-232, all of which relapsed despite the biological promise of this small molecule. By analyzing on-treatment biopsy samples, we detected upregulation of p53 transcriptional targets, suggesting blood-brain barrier penetrance and drug pharmacodynamics. However, in patient-derived cell lines, treatment with KRT-232 at the clinically achieved concentration was only sufficient to stall but not reduce tumor growth. Cell death via apoptosis was achieved in these cell line models when MDM2 inhibition was combined with the chemotherapeutic agent temozolomide. These drugs have non-overlapping mechanisms of action and exhibit synergy in our cell line models. Normal human bone marrow cells underwent a reversible growth inhibition phenotype when treated with KRT-232 and TMZ, supporting the idea that a therapeutic window exists with this combination. We additionally observed that tumor progression occurred in the absence of p53 inactivating mutations, suggesting that other mechanisms of resistance to KRT-232 may modulate drug response and resistance in GBM. Indeed, drug treatment induced an adaptive response in human tumors, characterized by the upregulation of genes involved in glial cell differentiation processes. Ongoing work is focused on understanding how tumor heterogeneity and cell state modulate response and resistance of GBMs to MDM2 inhibition and other therapeutic strategies that reactivate p53 signaling. Our study highlights the utility of tissue sampling during clinical trials and represents the first clinical effort to detect adaptive changes in human tumor tissue associated with p53 reactivation.

Dual IKZF2 and CK1α Degrader Targets Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive hematologic disease for which several epigenetic regulators have been identified as therapeutic targets. We previously found that a transcription factor and Ikaros family member, IKZF2 (HELIOS) is required for myeloid leukemic stem cell activity (Park et al. Cell Stem Cell 2019). Additionally, IKZF2 enhances self-renewal by increasing HOXA9 gene expression and in parallel blocks myeloid differentiation by reducing C/EBP expression. Thus, our studies suggested that targeting IKZF2 could be a new therapeutic strategy in AML. Immunomodulatory drugs (IMiDs; lenalidomide and others) have been used clinically to specifically target and degrade other Ikaros family members. However, these have been unable to target IKZF2. Utilizing a structure-guided approach, we developed a small molecule with low nanomolar degrader activity against IKZF2 (DEG-35). DEG-35 treatment in different AML cell lines induce apoptosis and differentiation with IC50 values of 5 ±0.4 nM in MOLM-13 cells to 27± 2.5 uM in NB4 cells. To understand if DEG35 has activity against other substrates, we performed global quantitative proteomics and found casein kinase 1 alpha (CK1α), a serine/threonine kinase previously identified to be critical for myeloid leukemogenesis and a known IMiD substrate, to be the most depleted protein (Jaras et al. JEM). Also, we performed a PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) screen assay which utilizes 770 barcoded cell lines from 20 lineages and identified multiple correlations with the p53 pathway. Previous studies found that CK1α suppresses the activity of p53 phosphorylation or binding MDM proteins. To further probe the mechanism behind DEG35 activity, we performed RNA sequencing at 24hrs in MOLM-13 cells. Gene set enrichment analysis (GSEA) using the rank list of differentially expressed genes from the MOLM-13 cells treated with DMSO and DEG-35 revealed enrichment for genes upregulated in CK1α KO keratinocytes and p53 targets. Additionally, we found enrichment for the MLL-AF9 IKZF2 KO LSC gene sets, myeloid differentiation, loss of MEIS1-HOXA9 and MYC targets. Immunoblotting validated the reduction of MYC and HOXA9 protein in DEG-35 treated MOLM-13 cells. To determine the contribution of CK1α and IKZF2 to the cellular phenotypes in MOLM13 cells, we developed CK1α and IKZF2 nondegradable mutants. CK1α, G40N was able to significantly reduce the cell killing activity of DEG-35 by 94% compared to vector cells (**p<0.01). Moreover, p53 deletion was also able to block DEG-35 induced apoptosis by 98% and differentiation by 88% compared to control. Additionally, expression of IKZF2 H141Q rescued DEG-35 mediated differentiation by 55% (***p<0.001), but not apoptosis. Thus, we demonstrate that the degradation of IKZF2 and CK1α by DEG-35 blocks cell growth and induces myeloid differentiation in human AML cell lines through CK1α-p53- and IKZF2-dependent pathways. To further probe the efficacy and therapeutic index of targeting both IKZF2 and CK1α. We found that AML cells were more sensitive to DEG-35 compared to normal bone marrow cells (murine MLL-AF9 Crbn I1391V cells has 10 fold and AML PDX cells have 2-5 fold higher sensitivity to normal mouse or human bone marrow cells).Treatment of DEG-35 at 50 mg/kg in AML PDX-transplanted mice led to significant prolonged survival compared to vehicle treated mice (median survival of DMSO mice: 45 days vs DEG-35 mice: 61 days; ***p< 0.001) and reduced disease burden. To improve pharmacological properties, we developed analog DEG-77, which has 10-fold increased solubility than DEG-35 in DMSO. DEG77 exhibited better in vivo efficacy in the MLL-AF9 Crbn I1391V model (Increase in median survival by DEG-35: 2 days *p<0.05 vs DEG-77: 31 days ****p<0.0001). Importantly, the increase of median survival of 30 days (****p<0.0001) was also observed in the secondarily transplanted DEG-77 mice compared to DMSO mice, suggesting that DEG-35 can reduce LSC activity. In summary, we report the development of nanomolar monofunctional cereblon-dependent degraders of IKZF2 and casein kinase 1 alpha (CK1α). DEG-35 or the analog DEG-77 delays leukemia progression in murine and human AML mice models. Thus, we provide a novel strategy for multi-targeted degradation of IKZF2/CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Interleukin-4 and Interleukin-5 Map to Human Chromosome 5 in a Region Encoding Growth Factors and Receptors and Are Deleted in Myeloid Leukemias With a del(5q)

Interleukin-4 (IL-4) is a potent mediator of growth and differentiation of cells of several hematopoietic lineages. Interleukin-5 (IL-5) is a lineage-specific hematopoietic growth factor that stimulates the production of eosinophils and eosinophil colonies from normal human bone marrow cells. By using somatic cell hybrids and in situ chromosomal hybridization, we localized the IL-4 and IL-5 genes to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, the IL-4 and IL-5 genes were found to be deleted in the 5q- chromosome of four patients with refractory anemia (RA) or therapy-related acute nonlymphocytic leukemia (t-ANLL), who had a del(5q). Thus a small segment of chromosome 5 contains IL-4, IL-5, IL-3, and GM-CSF as well as other genes such as CD14 and EGR1. Our findings that each of these genes was deleted in the 5q- chromosome suggest that loss of function of one or more of these genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q).

Paired Analyses of AML at Diagnosis and Relapse By Single-Cell RNA Sequencing Identifies Two Distinct Relapse Patterns

Introduction: Despite achievement of complete remission (CR) following chemotherapy, Acute Myelogenous Leukemia (AML) relapses in the majority of adult patients. While relapsed AML is almost always clonally related to the disease at diagnosis, the actual molecular and cellular contributors to chemotherapy resistance and to AML relapse remain incompletely understood. Some molecular determinants of relapse have been identified in genomic, epigenetic and proteomic aberrations, while cellular relapse reservoirs have been identified in leukemia stem cells as well as in more mature leukemic cell compartments. Here, we set out to determine the cellular composition, gene mutation status and gene expression of paired AML specimens procured at diagnosis and at relapse aiming at a better understanding of the AML relapse process. Methods: We employed the drop-seq 3' single cell RNA sequencing (scRNA-seq) method (Macosko 2015) with minor modifications to analyze the mRNA expression in single cells derived from 12 paired AML specimens procured at diagnosis and at relapse from prior CR. We obtained scRNA-seq data on 1000-2000 single cells per sample detecting approximately 2000-3000 unique molecular identifiers (UMIs) and 800-1500 genes per cell. Using WES or panel-based sequencing we determined mutations in known driver genes. Subsequently, we optimized novel methods for detection and mapping of mutated driver genes to individual cells using mutation specific PCR conditions and novel bioinformatics approaches. We annotated scRNA-seq expression profiles of the diagnosis and relapsed AML specimens individually using publicly available data for cell type-specific RNA markers derived from sorted normal cell populations and further compared the scRNA-seq data to scRNA-seq data of 5 pooled normal human bone marrows generated for this study. Results: Through analyses of scRNA-seq data of paired diagnosis and relapse AML specimens via principle components analyses (PCA) or t-distributed stochastic neighbor embedding (t-SNE) we detected varying degrees of separation of cell clusters in all cases analyzed indicative of substantial changes in single cell gene expression between AML diagnosis and relapse. A few of these observed cluster shifts were paralleled by gain or loss of mutated genes (e.g. FLT3-ITD) at relapse while most others lacked obvious clonal genomic markers. Through subsequent comparison of the expression similarities of single AML cells to sorted normal human bone marrow cells we detected two distinct AML relapse patterns: i) a pattern of relapse suggesting simple leukemia regrowth as evidenced by similar proportions of leukemia cells mapping onto discrete normal bone marrow cells (e.g. monocyte-like or GMPs or CMPs), and, ii) a pattern of relapse whereby the gene expression of relapsed cells (but not diagnosis cells) had similarity to normal hematopoietic cells that are conventionally placed more apical in the classical hematopoiesis differentiation cascade (HSCs, MPPs, CMPs; a phenotypic shift to immaturity). In addition, no leukemia sample mapped to just one classically defined bone marrow cell type but instead to multiple cell types, suggesting that most AML leukemia cells harbor aberrant hybrid cell gene expression patterns. Finally, we detected quantitative shifts in T cells and NK cells in some samples at relapse, which will be analyzed in greater detail. Conclusions: The comparative analysis of scRNA-seq data of paired AML specimens procured at diagnosis and relapse, identifies frequent and previously unrecognized changes in gene expression in leukemia cells at relapse. Through a comparison of gene mutation and gene expression at single cell resolution we identify two distinct AML relapse patterns in adult AML. Disclosures No relevant conflicts of interest to declare.

PS1234 APPLICATION OF FLOW SOM TO CLASSICAL FLOW CYTOMETRY: UNSUPERVISED ANALYSIS OF HEMATOPOIESIS AND HEMATOLOGICAL MALIGNANCIES

Background:Multiparameter flow cytometry (MFC) is an essential tool for the diagnosis and follow‐up of hematological malignancies. Numerous panels have been devised that best allow to achieve these goals. However, analysis of MFC graphs still heavily relies on the expertise of biologists and on specifically well adapted cascade‐gating strategies. Because of the development of mass cytometry, which generates data in a multidimensional environment of up to 50 dimensions, new strategies have been developed, that could be applied to classical MFC.Aims:In this study, the capacities of a rapid new tool for multidimensional analysis, FlowSOM (self‐organizing maps) have been combined to a classical MFC analysis tool, Kaluza®, for the identification of normal and diseased human bone marrow (BM) cell subpopulations.Methods:Normal BM samples collected during bone surgery were analyzed in MFC with four antibody panels adapted to the exploration of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) respectively. The listmode files obtained were merged and submitted to FlowSOM analysis, using R packages, which yielded trees of 100 nodes defining normal BM cell subsets. FlowSOM parameters were then transferred to Kaluza® for a bidimensional representation of the trees within which selected navigation could be performed with Kaluza® tools. In a second part of the study, the same stainings were performed on myelodysplastic or leukemic (AML or ALL) samples (diagnosis or follow‐up), which were submitted to FlowSOM together with the reference merged BM.Results:Node‐by‐node selection allowed for a precise definition of each of the unsupervised subsets generated by the software. This disclosed a selective segregation of delicate subsets such as non‐classical monocytes, hematogones or stem cells. Direct simultaneous comparison of diseased samples and normal BM allowed for a precise definition of abnormal cells (blasts) at diagnosis, and their immediate recognition as minimal residual disease in follow‐up samples.Summary/Conclusion:Flow SOM appears as a new and efficient tool for a rapid identification of BM cell subsets, highlighting differences from normal BM in dysplastic of leukemic samples. The node‐by‐node approach facilitates the definition of abnormal cells and their quantification, supervision intervening only after the generation of subsets delineated by artificial intelligence.

PS1235 RED BLOOD CELL GENERATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLS

Background: Multiparameter flow cytometry (MFC) is an essential tool for the diagnosis and follow-up of hematological malignancies. Numerous panels have been devised that best allow to achieve these goals. However, analysis of MFC graphs still heavily relies on the expertise of biologists and on specifically well adapted cascade-gating strategies. Because of the development of mass cytometry, which generates data in a multidimensional environment of up to 50 dimensions, new strategies have been developed, that could be applied to classical MFC. Aims: In this study, the capacities of a rapid new tool for multidimensional analysis, FlowSOM (self-organizing maps) have been combined to a classical MFC analysis tool, Kaluza®, for the identification of normal and diseased human bone marrow (BM) cell subpopulations. Methods: Normal BM samples collected during bone surgery were analyzed in MFC with four antibody panels adapted to the exploration of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) respectively. The listmode files obtained were merged and submitted to FlowSOM analysis, using R packages, which yielded trees of 100 nodes defining normal BM cell subsets. FlowSOM parameters were then transferred to Kaluza® for a bidimensional representation of the trees within which selected navigation could be performed with Kaluza® tools. In a second part of the study, the same stainings were performed on myelodysplastic or leukemic (AML or ALL) samples (diagnosis or follow-up), which were submitted to FlowSOM together with the reference merged BM. Results: Node-by-node selection allowed for a precise definition of each of the unsupervised subsets generated by the software. This disclosed a selective segregation of delicate subsets such as non-classical monocytes, hematogones or stem cells. Direct simultaneous comparison of diseased samples and normal BM allowed for a precise definition of abnormal cells (blasts) at diagnosis, and their immediate recognition as minimal residual disease in follow-up samples. Summary/Conclusion: Flow SOM appears as a new and efficient tool for a rapid identification of BM cell subsets, highlighting differences from normal BM in dysplastic of leukemic samples. The node-by-node approach facilitates the definition of abnormal cells and their quantification, supervision intervening only after the generation of subsets delineated by artificial intelligence.

Derepression of the Iroquois Homeodomain Transcription Factor Gene IRX3 Confers Differentiation Block in Acute Leukemia

SummaryThe Iroquois homeodomain transcription factor gene IRX3 is expressed in the developing nervous system, limb buds, and heart, and transcript levels specify obesity risk in humans. We now report a functional role for IRX3 in human acute leukemia. Although transcript levels are very low in normal human bone marrow cells, high IRX3 expression is found in ∼30% of patients with acute myeloid leukemia (AML), ∼50% with T-acute lymphoblastic leukemia, and ∼20% with B-acute lymphoblastic leukemia, frequently in association with high-level HOXA gene expression. Expression of IRX3 alone was sufficient to immortalize hematopoietic stem and progenitor cells (HSPCs) in myeloid culture and induce lymphoid leukemias in vivo. IRX3 knockdown induced terminal differentiation of AML cells. Combined IRX3 and Hoxa9 expression in murine HSPCs impeded normal T-progenitor differentiation in lymphoid culture and substantially enhanced the morphologic and phenotypic differentiation block of AML in myeloid leukemia transplantation experiments through suppression of a terminal myelomonocytic program. Likewise, in cases of primary human AML, high IRX3 expression is strongly associated with reduced myelomonocytic differentiation. Thus, tissue-inappropriate derepression of IRX3 contributes significantly to the block in differentiation, which is the pathognomonic feature of human acute leukemias.

Oxidative stress-induced JNK/AP-1 signaling is a major pathway involved in selective apoptosis of myelodysplastic syndrome cells by Withaferin-A.

Myelodysplastic syndromes (MDS) are a diverse group of malignant clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, dysplastic cell morphology in one or more hematopoietic lineages, and a risk of progression to acute myeloid leukemia (AML). Approximately 50% of MDS patients respond to current FDA-approved drug therapies but a majority of responders relapse within 2-3 years. There is therefore a compelling need to identify potential new therapies for MDS treatment. We utilized the MDS-L cell line to investigate the anticancer potential and mechanisms of action of a plant-derived compound, Withaferin A (WFA), in MDS. WFA was potently cytotoxic to MDS-L cells but had no significant effect on the viability of normal human primary bone marrow cells. WFA also significantly reduced engraftment of MDS-L cells in a xenotransplantation model. Through transcriptome analysis, we identified reactive oxygen species (ROS)-activated JNK/AP-1 signaling as a major pathway mediating apoptosis of MDS-L cells by WFA. We conclude that the molecular mechanism mediating selective cytotoxicity of WFA on MDS-L cells is strongly associated with induction of ROS. Therefore, pharmacologic manipulation of redox biology could be exploited as a selective therapeutic target in MDS.

T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death.

T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.

Presence of Primary Cilia in Human Hematopoietic System

Primary cilia are non-motile microtubule-based organelles that are present on the cellular membrane of all eukaryotic cells. The presence of cilia is required for the response to developmental signaling pathways such as Hedgehog (Hh) and Wnt/β-catenin. Loss of primary cilia or their function has been linked to a number of human genetic diseases, called ciliopathies (Dorn et al., Dev Cell, 2012). Recent studies suggest that abnormal hedgehog Hh pathway activity due to ciliary dysfunction could lead to various cancers such as glioblastoma, medulloblastoma, basal cell carcinoma, breast, prostate and ovarian cancer (Menzl et al., Cilia, 2014, Moser et al., BMC cancer 2009). Although the Hh pathway has emerged as an exciting new therapeutic target in leukemia and myeloproliferative diseases, to date, there have been no reports describing the presence of primary cilia in human blood or leukemia cells. In the present study, we have successfully identified primary cilia in hematopoietic system (Fig. 1). Primary cilia are well characterized in hedgehog responsive NIH3T3 cells; therefore, these cells were included in the study as a positive control for cilia staining. Localization of acetylated tubulin (which stains for the ciliary body) and gamma tubulin (which stains the basal body) was used to confirm the presence of primary cilia in normal human peripheral blood mononuclear cells (PBMCs) and normal bone marrow (BM) cells. Furthermore, we have also identified primary cilia in acute myeloid leukemia cell lines (KG1, KG1a, K562). Additionally, hedgehog pathway components such as, Smoothened and Gli3 were localized in primary cilia of acute myeloid leukemia (AML) cell lines. Interestingly, the frequency of primary cilia in AML cells (10-36%) was found to be lower than normal PBMCs and BM (97-99%) (Fig. 1). Moreover, cilia present on leukemic cells were often shorter and dysmorphic, with less well defined basal bodies. KG1 cells, which have a higher number of cilia, retain sensitivity to Hh pathway inhibitors, but K562 and KG1a, which have high levels of Hh activity but very low numbers of cilia, are resistant. Loss of cilia is, therefore, associated with activation of Hh signaling that is resistant to SMO antagonists, and could have important implications for the development of this class of drugs. In conclusion, we present the first evidence showing the presence of primary cilia in hematologic system and implicate the loss of primary cilia in development of hematological malignancies through deregulated Hh signaling. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ONC201 Exerts p53-Independent Cytotoxicity Through TRAIL and DR5 Induction In Mantle Cell Lymphomas

ONC201 (TIC10) is a novel small molecule that induces TRAIL-dependent apoptosis in various cancer cell types and is under development to enter a first-in-man study in advanced cancer patients .It was identified in a screen for small molecules capable of up-regulating endogenous TRAIL gene transcription in a p53-independent manner (Allen JE et al, Sci Transl Med., 2013). ONC201 triggers FOXO3a activation through dual inhibition of ERK and AKT, which transcriptionally upregulates TRAIL and TNFRSF10B (TRAIL-R2/DR5) in solid tumors. Because PI3K/AKT and MEK/ERK activation have been shown to be major contributors to drug resistance, ONC201 is potentially promising since it not only promotes TRAIL activation, but also upregulates its pro-apoptotic receptor DR5.Here we report the anti-lymphoma effects of ONC201 in MCL, a presently incurable disease. We treated three human MCL cell lines with wild-type p53 (Z-138, JVM-2, and Granta-519) and two similar lines with mutant p53 (MINO and Jeko-1) with ONC201. A 72-hour ONC201 treatment induced apoptosis in all MCL cell lines. Surprisingly, the p53 mutant MINO and Jeko-1 cells were more susceptible in apoptosis assays to ONC201 than cells with wild-type p53 (Fig.1) The effective concentrations inducing cell killing (as measured by annexin V positivity) in 50%/75% of the cells in the Z-138, JVM-2, MINO, Jeko-1, and Granta-519 cells were 9.9/>10, >10/>10, 2.6/5.2, 2.7/4.6 and >10/ >10 micromolar, respectively. We also treated five primary human MCL samples (three with wild-type p53 and two with mutant p53), and found that one of the two mutant p53 samples was highly sensitive to ONC201 as were the three samples with wild-type p53. One mutant p53 sample that was less sensitive to ONC201, was also resistant to Nutlin-3a and Ibrutinib suggesting an extremely drug-resistant phenotype. Real-time PCR analysis revealed that both DR5 and TRAIL mRNAs were transcriptionally upregulated in the primary MCL samples (a relative ratio of 7.25 compared to 3.13 in controls) after 72-hour treatment with ONC201. [Display omitted] To determine the significance of p53 functional status in ONC201-induced apoptosis, p53 wild-type Z-138 and JVM-2 cells were stably transduced with lentivirus encoding either negative control shRNA or p53-specific shRNA and were exposed to ONC201 and results demonstrated complete p53-independence. Normal human bone marrow cells and mesenchymal stem cells were completely resistant to the cytotoxic effects of ONC201, which illustrated this agent's low toxicity against normal tissues.In order to examine the role of p53 activation in ONC201-induced apoptosis in MCL cells, we combined ONC201 with the MDM2 inhibitor Nutlin-3a. The combination cytotoxic effects of this combination were synergistic in p53 wild-type Z-138 and JVM-2 cells (combination index 0.87 and 0.63, respectively). Similar synergistic effects of ONC201 combined with the BTK inhibitor Ibrutinib were observed in Z-138 and MINO cells (combination index 0.63 and 0.61, respectively). This combination also triggered synergistic apoptotic effects in two primary MCL samples with combination indexes of 0.0011 and 0.073, respectively. ConclusionONC201 induces p53-independent apoptosis in MCL cells, and may have significant clinical impact by targeting both p53 wild type and p53 mutant drug-resistant MCL cells. ONC201 exerts synergistic effects with MDM2 and BTK inhibitors that may be explored clinically. Disclosures:Allen:Drug Company: Employment. Andreeff:Oncoceutics: SAB Other.

PRL2 Maintains Hematopoietic Stem and Progenitor Cells Through Regulating SCF/KIT Signaling

The phosphatase of regenerating liver family of phosphatases, consisting of PRL1, PRL2 and PRL3, represents an intriguing group of proteins implicated in cell proliferation and tumorigenesis. However, the role of PRLs in normal and malignant hematopoiesis is largely unknown. While SCF/KIT signaling plays an important role in hematopoietic stem and progenitor cell (HSPC) maintenance, how SCF/KIT signaling is regulated in HSPCs remains poorly understood. We here report that PRL2 regulates HSPC maintenance through regulating SCF/KIT signaling. To define the role of PRL2 in hematopoiesis, we analyzed the hematopoietic stem cell (HSC) behavior in Prl2 deficient mice generated by our group. Prl2 deficiency results in ineffective hematopoiesis and impairs the long-term repopulating ability of HSCs. In addition, Prl2 null HSPCs are less proliferative and show decreased colony formation in response to SCF stimulation. Furthermore, Prl2 null HSPCs show reduced activation of the PI3K/AKT and ERK signaling in steady state and following SCF stimulation. Importantly, we found that PRL2 associates with KIT and the ability of PRL2 to enhance SCF signaling depends on its enzymatic activity, demonstrating that PRL2 mediates SCF/KIT signaling in HSPCs. Thus, PRL2 plays a critical role in hematopoietic stem and progenitor cell maintenance through regulating SCF/KIT signaling. Furthermore, loss of Prl2 decreased the ability of oncogenic KITD814V mutant in promoting hematopoietic progenitor cell proliferation and in activation of signaling pathways. We also checked the expression of PRL2 proteins in human AML cell lines and found increased level of PRL2 proteins in some acute myeloid leukemia (AML) cells compared with normal human bone marrow cells, indicating that PRL2 may play a pathological role in AML. Our results suggest that the PRL2 phosphatase may be a druggable target in myeloproliferative disease (MPD) and acute myeloid leukemia (AML) with oncogenic KIT mutations. Disclosures:No relevant conflicts of interest to declare.

OSU-A9, An Indole-3-Carbinol Derivative, Induces Cytotoxicity In Acute Myeloid Leukemia Through Reactive Oxygen Species-Mediated Apoptosis

IntroductionIndole-3-carbinol (I3C) is a broadly targeted phytochemical shown to prevent carcinogenesis in animal studies and to suppress the proliferation of cancer cells of human breast, colon, prostate, and endometrium. Here we aim to test the anticancer effect of OSU-A9, an I3C derivative with improved potency, in acute myeloid leukemia (AML). Materials and MethodsThe in vitro activity of OSU-A9 was evaluated in AML cell lines (HL-60 and THP-1) and primary leukemia cells from 18 AML patients. THP-1 xenograft tumors in athymic nude mice was used for in vivo study. ResultsOSU-A9 mediates cytotoxicity in AML cell lines and primary leukemia cells from AML patients in a dose-responsive manner. The IC50 at 24 h for 18 patients was 1.63 μM. Normal human bone marrow cells were much less sensitive to OSU-A9 with an IC50 at 24 h greater than 8 μM. OSU-A9 causes cytotoxicity dependent on caspase activation, as evidenced by caspase-3 and PARP cleavage, and induces autophagy but not autophagic cell death. Interestingly, pretreatment of AML cell lines and primary AML cells with N-acetylcysteine or glutathione rescues them from apoptosis (and concomitant PARP cleavage) and Akt hypophosphorylation, implicating a key role of reactive oxygen species (ROS) in OSU-A9-related cytotoxicity. To investigate the anti-leukemia effect of OSU-A9 in vivo, fifteen male athymic nude mice were xenografted with THP-1 cells. Briefly, the anticancer utility of OSU-A9 is extended in vivo as it, administered intraperitoneally, suppresses the growth of THP-1 xenograft tumors in athymic nude mice without obvious toxicity. For biomarker analysis in the THP-1 xenografts, protein extracts were obtained from the tumors and immunoblotted for Akt levels. The tumors from OSU-A9 treated mice exhibited down regulation of Akt phosphorylation compared with those from placebo-controlled mice. ConclusionsThis study shows that ROS-mediated apoptosis contributes to the anticancer activity of OSU-A9 in AML cell lines and primary AML cells, and thus should be considered in the future assessment of its translational value in AML therapy. Disclosures:No relevant conflicts of interest to declare.

OSU-A9, an indole-3-carbinol derivative, induces cytotoxicity in acute myeloid leukemia through reactive oxygen species-mediated apoptosis

Indole-3-carbinol (I3C) is a broadly targeted phytochemical shown to prevent carcinogenesis in animal studies and to suppress the proliferation of cancer cells of human breast, colon, prostate, and endometrium. Here we demonstrate that OSU-A9, an I3C derivative with improved anticancer potency, induces cytotoxicity in acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells were less sensitive to OSU-A9 than leukemia cells. OSU-A9 induces caspase activation, PARP cleavage, and autophagy but not autophagic cell death. Interestingly, pretreatment of AML cell lines and primary AML cells with N-acetylcysteine or glutathione rescues them from apoptosis (and concomitant PARP cleavage) and Akt hypophosphorylation, implicating a key role of reactive oxygen species (ROS) in OSU-A9-related cytotoxicity. Importantly, the anticancer utility of OSU-A9 is extended in vivo as it, administered intraperitoneally, suppresses the growth of THP-1 xenograft tumors in athymic nude mice without obvious toxicity. This study shows that ROS-mediated apoptosis contributes to the anticancer activity of OSU-A9 in AML cell lines and primary AML cells, and thus should be considered in the future assessment of its translational value in AML therapy.

Identification of the Wnt Signalling Protein, TCF7L2 As a Significantly Overexpressed Transcription Factor in AML

AbstractAbstract 1281Acute Myeloid Leukaemia (AML) is one of the most genetically heterogeneous malignancies with the defining feature of inhibition of terminal myeloid development arising from a disordered development programme. To discover the most commonly dysregulated genes in this disease we compared Affymetrix GC-RMA normalized mRNA expression data of normal human hematopoietic progenitor (CD34+) cells with diagnostic samples from 223 AML patients previously enrolled in AML NCRI-MRC UK Trials. We used Mixed Model ANOVA and MetaCore™ to identify dysregulated gene networks. Processes associated with Wnt signalling were found to be the most significantly dysregulated: Wnt and cytoskeletal remodelling (p=1.16−26) and; developmental Wnt signalling (p=2.91−16). Interactome analysis identified TCF7L2 (aka TCF4) as the most significantly dysregulated Wnt factor (p=7.56−25; z-score = 19.87) which showed elevated expression in 78 percent of AML patients. The TCF7L2/β-catenin complex is a nuclear regulator of canonical Wnt signalling that is involved in the development of several cancers. To validate this result we examined the expression of TCF7L2 by RQ-PCR using matched AML samples (n=13). These data significantly correlated with the normalized array expression data (R= 0.711; p=0.006) which in turn correlated with total protein expression determined by Western blotting (n=16);R=0.598; p= 0.014 (see also below). To gain further insights into the prognostic impact of TCF7L2 expression at diagnosis in AML, we next examined a number a variables including patients’ age, leukocytes count, prognostic group, gender, WHO performance in multivariate analysis. We have shown that TCF7L2 over expression was found to be independently prognostic for reduced complete remission rate after the adjustment for these co-variables (p=0.0144), OR=5.19 [95% C.I.=1.39–19.39].The TCF7L2 gene undergoes exon splicing which yields multiple isoforms which have been reported to yield functionally distinct proteins. We therefore compared TCF7L2 mRNA isoform expression in AML patients with normal human bone marrow and progenitor cells. As expected we found extensive variability of TCF7L2 splicing particularly at the 3’ end of the gene (exon 13–18). Bone marrow and progenitor cells isoforms had distinct exon composition (with respect to exon 4) suggesting developmental regulation of isoform expression. AML patients were heterogeneous in the isoform expression pattern, but aberrant exon composition (compared to normal cells) was not observed. At the protein level we detected expression of 72kDa (corresponding to predicted exon composition 1–15 and 18) and 58kDa (exons 1–13 and 18) protein isoforms. In both normal and AML cells, expression of the 58kDa form was dominant.To determine the functional significance of TCF7L2 overexpression we employed lentiviral shRNA vectors targeting TCF7L2 isoforms in leukemic cell lines (K562 and U937) engineered to co-express a TCF-GFP reporter which enabled simultaneous analysis of the effect on TCF-dependent transcription. TCF reporter activity was inhibited by shRNA expression and the cells became non-responsive to Wnt agonists (Wnt3A and BIO) demonstrating that canonical Wnt signalling is dependent on TCF7L2 expression in these cells. Phenotypically, shRNA expression was found to strongly inhibit proliferation and to promote apoptosis indicating that TCF7L2 expression is required for the proliferation and survival of myeloid leukemia cells. In summary these data identify Wnt signalling as the most commonly dysregulated process in AML and show that one of key mediator of canonical Wnt signalling, TCF7L2, is overexpressed in AML, is associated with poor clinical outcome, and is functionally required to maintain the proliferation and viability of myeloid leukemia cells. Disclosures:No relevant conflicts of interest to declare.

PDCD2 knockdown inhibits erythroid but not megakaryocytic lineage differentiation of human hematopoietic stem/progenitor cells

Programmed cell death-2 (PDCD2) protein is enriched in embryonic, hematopoietic, and neural stem cells, however, its function in stem/progenitor cell differentiation is unclear. We investigated the effects of PDCD2 knockdown on the development and differentiation of hematopoietic progenitor cells (HPC). CD34(+) cells derived from normal human bone marrow and K562 leukemic cells were effectively transduced with short-hairpin RNA to knockdown PDCD2. Colony-forming assays were used to investigate the effects of PDCD2 loss on HPC clonogenic potential and on 12-O-tetradecanoyl-phorbol-13-acetate-and arabinofuranosylcytosine-induced terminal differentiation. In CD34(+) clonogenic progenitors, PDCD2 knockdown decreased the total number of colony-forming units, increased the number of colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte and burst-forming unit-erythroid primitive colonies, and decreased the number of burst-forming unit-erythroid mature colonies. Similar results were observed in K562 cells, suggesting that PDCD2 is important for HPC differentiation and/or survival, and for erythroid lineage commitment. Furthermore, 12-O-tetradecanoyl-phorbol-13-acetate-induced megakaryocytic differentiation and proliferation of K562 cells was not affected by PDCD2 knockdown. In contrast, arabinofuranosylcytosine-induced erythroid differentiation of K562 cells was significantly reduced with PDCD2 knockdown, with no effect on cell proliferation. The effects of PDCD2 knockdown were attributed to a cell cycle arrest at G(0)/G(1), along with increased messenger RNA expression of early progenitor factors c-MYB and GATA-2, and decreased expression of erythroid factors GATA-1, EpoR, and γ-globin. We conclude that PDCD2 loss of function(s) impedes erythroid differentiation by inducing cell cycle arrest and increasing expression of early hematopoietic progenitor factors. These findings suggest that PDCD2 has a novel regulatory role in human hematopoiesis and is essential for erythroid development.

Distribution and shedding of the membrane phosphatidylserine during maturation and aging of erythroid cells

Maturation and aging of erythroid cells are accompanied by extensive remodeling of the membrane and a marked decrease in cell size, processes that are mediated by externalization and shedding of phosphatidylserine (PS). In the present study, we investigated the redistribution of PS in the plasma membrane of erythroid precursors during their maturation and of mature RBCs during senescence, and the involvement of changes in calcium (Ca)-flux in these processes. Maturation was studied by analyzing normal human bone marrow cells as well as cultured human normal erythroid precursors induced by erythropoietin and murine erythroleukemia cells induced by hexamethylene-bisacetamide. Senescence was studied in normal human peripheral RBCs following density fractionation. PS and Ca were determined by flow cytometry using annexin-V and Flu-3, respectively. The outer, inner and shed PS were quantified by a novel two-step binding inhibitory assay. The results indicate a bi-phasic modulation of intracellular Ca and PS externalization/shedding; both of which decreased during maturation and increased during aging. The role of intracellular Ca in PS externalization/shedding was demonstrated by modulating intracellular Ca: Ca was decreased by incubating the cells with an ion chelator (EDTA) or with decreasing concentrations of Ca, whereas treatment with the ionophore A23187 elevated intracellular Ca. The results showed that low Ca resulted in decreased outer and shed PS, whereas high Ca had the opposite effect. The results suggest that PS externalization and shedding are mediated by increased cellular Ca-flux, and that they play an important role in erythroid maturation and RBC senescence.

Inhibitory effect of phenylbutazone and oxyphenbutazone on DNA synthesis in normal human bone marrow cells in vitro

Inhibitory effect of phenylbutazone and oxyphenbutazone on DNA synthesis in normal human bone marrow cells <i>in vitro</i>

Microvesicle Induction of Prostate Specific Gene Expression in Normal Human Bone Marrow Cells

Transfer of genetic material from cancer cells to normal cells occurs via microvesicles. Cell specific phenotypes can be induced in normal cells by the transfer of material in microvesicles, leading to genetic changes. We report the identification and expression of prostate specific genes in normal human marrow cells co-cultured with human prostate cancer cells. We harvested prostate tissue from 11 patients with prostate cancer. In 4 cases prostate tissue was co-cultured across from human marrow for 2 or 7 days but separated from it by a 0.4 μM polystyrene membrane. In 5 cases conditioned medium from patient cancer tissue was collected and ultracentrifuged, and microvesicles were collected for co-culture (3) and vesicle characterization (3). Explanted human marrow was harvested from cultures and RNA extracted. Real-time reverse transcriptase-polymerase chain reaction was done for select prostate specific genes. Marrow exposed to human prostate tumor or isolated microvesicles in culture in 4 and 3 cases, respectively, showed at least 2-fold or greater prostate gene expression than control marrow. In 1 case in which normal prostate was co-cultured there were no prostate gene increases in normal marrow. Prostate cancer tumor cells co-cultured with human bone marrow cells induce prostate specific gene expression. The proposed mechanism of transfer of genetic material is via microvesicles. This represents an opportunity for novel therapeutic agents, such as antibodies, to block microvesicle release from cancer cells or for agents that may block cells from accepting microvesicles.