The Correlation Between Children’s Acute Lymphoblastic Leukemia Drug Resistance System Induced by Metabolomics-Based 6-Mercaptopurine and Hypoxanthine-Guanine Phosphoribosyl Transferase 1 Protein

This study aimed to explore 6-mercaptopurine (MP)-induced children’s acute lymphoblastic leukemia (ALL) drug resistance system and leukemia hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1) protein. Based on metabonomics, drug resistance of 6MP-Reh cell line was established by increasing concentration administration method, and the degree of drug resistance of 6MP-Reh was verified by apoptosis test, western blotting (WB) test, and drug sensitivity test. The changes of tissue inhibitor of matrix metalloproteinase (TIMP) and thioguanosine monophosphate (TGMP) in drug-resistant cells were detected through liquid chromatograph (LC)/mass spectrometer (MS). The 6MP-Reh-wt cell line was established by lentivirus infection, so as to verify the correlation between HPRT1 and drug resistance mechanism. The results showed that the inhibition concentration (IC50) value, cell vitality (CV), apoptosis rate, and 6-MP content of 6MP-Reh were higher hugely than those of Reh (P < 0.05). The contents of HPRT1, TIMP, and TGMP in 6MP-Reh cells were lower sharply than the contents of Reh cells (P < 0.001). The IC50 value of 6MP-Reh-wt was also lower steeply than the value of 6MP-Reh (P < 0.001), and the concentrations of TIMP and TGMP increased obviously (P < 0.05). Therefore, it indicated that the mutation of HPRT1 in drugresistant cell lines could lead to a decrease in their viability and cause leukemia cells to develop resistance to 6-MP. In addition, HPRT1 gene could improve their resistance to 6-MP.

Autophagy Regulates VDAC3 Ubiquitination by FBXW7 to Promote Erastin-Induced Ferroptosis in Acute Lymphoblastic Leukemia

Background: The discovery of ferroptosis is a major breakthrough in the development of cancer treatments. However, the mechanism by which ferroptosis contributes to acute lymphoblastic leukemia (ALL) is to be clarified. Here, we explored erastin-induced ferroptosis in ALL cells and the impact of autophagic activity on this process.Materials and Methods: Cell viability was evaluated in various ALL cell lines following erastin treatment by the MTS assay, while cell death was evaluated via a trypan blue assay. Immunoblotting and quantitative real-time PCR were used to detect protein and mRNA expression, respectively. The UbiBrowser database was used to predict the E3 ligase of VDAC3, which was confirmed by immunoprecipitation. The role of FBXW7 in erastin-induced ferroptosis in vitro was evaluated via lentiviral-mediated silencing and overexpression. ALL xenograft mice were used to observe the impact of autophagy on erastin-induced ferroptosis.Results: Resistance to erastin-induced ferroptosis was higher in Jurkat and CCRF-CEM cells than in Reh cells. The sensitivity could be modified by the autophagy activator rapamycin (Rapa) and the autophagy inhibitor chloroquine (CQ). Rapa sensitized ALL cells to erastin-induced ferroptosis. In ALL xenograft mice, the combination treatment of Rapa and erastin resulted in longer survival time than those observed with erastin or Rapa treatment alone. VDAC3 was regulated by autophagy post-transcriptionally, mainly via the ubiquitin-proteasome system (UPS). FBXW7 was verified as a specific E3 ligase of VDAC3. FBXW7 knockdown attenuated VDAC3 degradation by suppressing its ubiquitination, thereby increasing the sensitivity of ALL cells to erastin.Conclusion: Autophagy regulated erastin-induced ferroptosis via the FBXW7-VDAC3 axis. Rapa sensitized ALL cells to erastin-induced ferroptosis both in vitro and in vivo. Our findings provide potential therapeutic targets for ALL.

Glucocorticoid-Resistant B-Cell Acute Lymphoblastic Leukemic Cells Can be Targeted By BCR-Signaling Inhibition

Glucocorticoids (GCs) remain a backbone component of therapeutic regimens for childhood B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is a strong prognostic marker of relapse making its understanding an important challenge to be addressed to improve overall patients outcome. Healthy B-cell development is characterized by checkpoints where critical regulatory signaling influences the fate of developing B-cells. These stages are vulnerable for leukemic transformation and as we previously demonstrated, the developmental and functional state of B-ALL cells are of critical importance in treatment failure. Using this developmental framework, we examined the effect of GCs on healthy and malignant B-cells to better understand mediators of GC resistance and ways to overcome it. To define the dynamics of the transcriptional networks surrounding B-cell developmental checkpoints, we performed transcriptomic analysis of sorted pre-proB, pro-B and pre-B cells from 3 healthy donors. We found a coordinated upregulation of B cell receptor (BCR) and Glucocorticoid Receptor (GCR) pathways in healthy B cells during their development. Single cell proteomic analysis of these healthy populations confirmed the coordinated expression of GCR with early B-cell phenotypic markers. Furthermore, in vitro treatment of healthy B-cells with the GC dexamethasone (dex) demonstrated cycling pro-B/pre-B cells to be the most sensitive to GC-induced cell death. Given the importance of GCs in B-ALL treatment, we investigated GCs effects and resistance in NALM6 and REH cell lines. NALM6, expressing high levels of GCR, were significantly sensitive to dex treatment in terms of cell cycle arrest and cell death. By contrast, REH cells were resistant to dex treatment as they lack the GCR. Retroviral transfection of NR3C1 (GCR gene) in REH cells (REH GCR) resulted in acquired sensitivity to dex. To explore the potential crosstalk between GCR and BCR pathways, we also treated cells with BCR signaling inhibitor, dasatinib (das), alone or in combination with dex. While both cell lines survived to treatment with das, the combined treatment increased apoptosis compared to dex alone in NALM6 cells (p=0.0179) and REH GCR cells (p=ns). Whole transcriptome sequencing of dex-resistant cells revealed upregulation of BCR downstream signaling as one of the main pathways associated with resistance. The cell line data implicated active BCR signaling as a path to GCs resistance, so we next analyzed 19 B-ALL primary samples by mass cytometry, after in vitro exposure for 48 hrs to same treatments. Across the entire cohort, dex induced a significant reduction in viability (p=0.0007), compared to vehicle. Treatment with das and dex+das also decreased cell viability compared to vehicle (p=0.0038 and p=0.0002) although was not significant to dex alone. Interestingly dex-resistant cells showed a phenotypic modulation compatible with a late pre-B phenotype with increased CD45 and CD20 expression. In addition, surviving cells showed an activation of downstream targets of BCR signaling such as pSYK, pRPS6 and pCREB that was partially blunted by dasatinib treatment. To understand whether the phenotype and signaling modulation induced by dexamethasone also occurs in patients after treatment with GCs, we analyzed minimal residual disease (MRD) cells following 8 days of treatment with GCs from 9 B-ALL patients. This analysis confirmed our previous findings with MRD cells having same late pre-B cells phenotype and signaling profile as in vitro treated cells. Finally, we tested whether targeting of pre-BCR signaling via dasatinib, could overcome resistance in vivo. We evaluated engraftment of luciferase-expressing NALM6 cells at different timepoints after tail vein injection in mice treated with vehicle, dex, das or dex + das. Bioluminescence analysis revealed a significant reduction of early and late engraftment in the dex + das group compared to vehicle-treated mice. Furthermore, mice receiving the combined treatment also experienced a significant survival advantage as assessed by log rank test (p=0.0002). Taken together these data suggest a coordinated interplay between BCR and GCR pathways in healthy and leukemic B cells. GCs-resistant leukemic cells showed a mature pre-B phenotype that is vulnerable to BCR signaling inhibition in vitro and in vivo suggesting new therapeutic options to overcome GC resistance in childhood B-ALL. Disclosures Biondi: Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria. Bava: 10x Genomics: Current Employment. Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

Knockdown of GPSM1 Inhibits the Proliferation and Promotes the Apoptosis of B-Cell Acute Lymphoblastic Leukemia Cells by Suppressing the ADCY6-RAPGEF3-JNK Signaling Pathway

B-cell acute lymphoblastic leukemia (B-ALL) is the common type of blood cancer. Although the remission rate has increased, the current treatment options for B-ALL are usually related to adverse reactions and recurrence, so it is necessary to find other treatment options. G protein signaling modulator 1 (GPSM1) is one of several factors that affect the basic activity of the G protein signaling system, but its role in B-ALL has not yet been clarified. In this study, we analyzed the expression of GPSM1 in the Oncomine database and found that the GPSM1 levels were higher in B-ALL cells than in peripheral blood mononuclear cells (PBMCs). Analyses of the Gene Expression Profiling Interactive Analysis (GEPIA) demonstrated that patients with high GPSM1 levels had shorter survival times than those with low levels. Additionally, gene set enrichment analysis (GSEA) suggested that GPSM1 was positively correlated with proliferation, G protein-coupled receptor (GPCR) ligand binding, Gαs signaling and calcium signaling pathways. In further experiments, GPSM1 was found to be highly expressed in Acute lymphoblastic leukemia (ALL) cell lines, and downregulation of GPSM1 inhibited proliferation and promoted cell cycle arrest and apoptosis in BALL-1 and Reh cells. Moreover, knockdown of GPSM1 suppressed ADCY6 and RAPGEF3 expression in BALL-1 and Reh cells. Furthermore, we reported that GPSM1 regulated JNK expression via ADCY6-RAPGEF3. The present study demonstrates that GPSM1 promotes tumor growth in BALL-1 and Reh cells by modulating ADCY6-RAPGEF3-JNK signaling.

THU0036 FIRST-IN-HUMAN TRIAL OF BCMA-CD19 COMPOUND CAR IN THE TREATMENT OF AUTOANTIBODY MEDIATED DISORDERS

Background:Donor-specific anti-HLA antibodies (DSAs) are antibodies in the recipient directed against donor class I/II HLA antigens. The existence of DSAs before allogenic hematopoietic stem cell transplantation (AHSCT) are known to cause primary graft failure. Currently there’s no established method of DSA desensitization due to the long half-life of plasma cells.Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease involving in multiple organ systems mediated by numerous autoantibodies. Recent results have shown that depletion of B cells by CD19 CAR-T cells effectively reversed some manifestations in two SLE mouse models. However, plasma cells could be spared with single CD19 CAR-T cells, and peripheral circulating anti-DNA IgG and IgM autoantibodies remain elevated or increased in treated mice.Objectives:We present the efficacy of BCMA-CD19 compound CAR (cCAR), which target on antibody- producing “root”, both B cells and plasma cells in preclinical study and in our first-in-human phase 1 clinical trial.Methods:We constructed a BCMA-CD19 cCAR composed of a complete BCMA-CAR fused to a complete CD19 CAR, separated by a self-cleaving P2A peptide. We assessed the functional activity of cCAR in co-culture assay with multiple cell lines. We also verified cCAR efficacy with two mouse models, injected with either BCMA-expressing MM.1S cells or CD19-expressing REH cells. In our phase 1 clinical trial, we enrolled patients with hematologic malignancies with antibody mediated disorders.Results:BCMA-CD19 cCAR exhibited robust cytotoxic activity against the K562 cells engineered to express either CD19 or BCMA in co-culture assays, indicating the ability of each complete CAR domain to specifically lyse target cells. In mouse model study, cCAR-T cells were able to eliminate tumor cells in mice injected with MM.1S cells and REH cells, indicating that both BCMA and CD19 are specifically and equally lysing B cells and plasma cells in vivo, making BCMA-CD19 cCAR a candidate for clinical use.In our first-in-human clinical trial, the first case is a 48-year-old female patient having resistant B-ALL with high DSA titers. She exhibited complete remission of B-ALL at day 14 post-CAR T treatment. MFI of DSA dropped from 7800 to 1400 at 8 weeks post cCAR treatment, the reduction percentage was approximately 80% (Figure 1). The patient had no CRS, and no neurotoxicity was observed.Figure 1.1. A) MFI of DSA and other HLA antibodies before and at different time points after cCAR T infusion. B) the percent reduction post-transfusion of cCAR T cells at different time points.The second case is a 41-year-old female patient having a refractory diffuse large B cell lymphoma with bone marrow (BM) involvement. Furthermore, she has a 20 years of SLE, with manifestation of fever dependent of corticosteroids. On day 28 after cCAR treatment, PET/CT scan showed CR, and BM turned negative. In addition, she is independent of steroids, has no fever and other manifestations, C3/C4 are within normal ranges, and all the ANA dropped significantly, especially the nuclear type ANA, which turned from> 1:1000 to be negative at day 64. She had Grade 1 CRS but with no neurotoxicity observed. The absence of B cells and plasma cells persisted more than 5 months post CAR therapy.Conclusion:Our first in human clinical trial on BCMA-CD19 cCAR demonstrated profound efficacy in reducing DSA levels in an AHSCT candidate and ANA titer in a SLE patient. There was strong clinical evidence of depletion of antibody-producing roots, B-cells and plasma cells in both patients. Our results further suggested that BCMA-CD19 cCAR has the potential to benefit patients receiving solid organ transplants or those with other antibody-mediated diseases.Figure 2.Reduction of different type of ANA titer at different time points.Acknowledgments:patients and their familiesDisclosure of Interests:Fang liu: None declared, Hongyu Zhang: None declared, Xiao Wang: None declared, Jia Feng: None declared, Yuanzhen cao Employee of: Employee of iCell Gene Therapeutics LLC, Yi Su: None declared, Masayuki Wada Employee of: employee of iCell Gene Therapeutics LLC, Yu Ma Employee of: employee of iCAR Bio Therapeutics Ltd, Yupo Ma Shareholder of: shareholder of iCell Gene Therapeutics LLC

High Mobility Group A1 Chromatin Regulators Function As Critical Drivers of Leukemogenesis in Preclinical Models of Relapsed, Pediatric B-Cell ALL

Introduction: Acute B-cell lymphoblastic leukemia (B-ALL) is the most common form of childhood leukemia and the leading cause of death in children with cancer. While therapy is often curative, about 10-15% of children will relapse with recurrent disease and abysmal outcomes. Actionable mechanisms that mediate relapse remain largely unknown. The gene encoding the High Mobility Group A1(HMGA1) chromatin regulator is overexpressed in diverse malignancies where high levels portend poor outcomes. In murine models, we discovered thatHmga1 overexpression is sufficient for clonal expansion and progression to aggressive acute lymphoid leukemia (Cancer Res 2008,68:10121, 2018,78:1890; Nature Comm 2017,8:15008). Further, HMGA1 is overexpressed in pediatric B-ALL (pB-ALL) blasts with highest levels in children who relapse early compared to those who achieve chronic remissions. Together, these findings suggest that HMGA1 is required for leukemogenesis and may foster relapse in B-ALL. We therefore sought to: 1) test the hypothesis that HMGA1 is a key epigenetic regulator required for leukemogenesis and relapse in pB-ALL, and, 2) elucidate targetable mechanisms mediated by HMGA1 in leukemogenesis. Methods: We silenced HMGA1 via lentiviral delivery of short hairpin RNAs targeting 2 different sequences in cell lines derived from relapsed pB-ALL (REH, 697). REH cells harbor the TEL-AML1 fusion; 697 cells express BCL2, BCL3, and cMYC. Next, we assessed leukemogenic phenotypes in vitro (proliferation, cell cycle progression, apoptosis, and clonogenicity) and leukemogenesis invivo. To dissect molecular mechanisms underlying HMGA1, we performed RNA-Seq and applied in silico pathway analysis. Results: There is abundant HMGA1 mRNA and protein in both pB-ALL cell lines and HMGA1 was effectively silenced by short hairpin RNA. Further, silencing HMGA1 dramatically halts proliferation in both cell lines, leading to a decrease in cells in S phase with a concurrent increase in G0/S1. Apoptosis also increased by 5-10% after HMGA1 silencing based on flow cytometry for Annexin V. In colony forming assays, silencing HMGA1 impaired clonogenicity in both pB-ALL cell lines. To assess HMGA1 function in leukemogenesis in vivo, we implanted control pB-ALL cells (transduced with control lentivirus) or those with HMGA1 silencing via tail vein injection into immunosuppressed mice (NOD/SCID/IL2 receptor γ). All mice receiving control REH cells succumbed to leukemia with a median survival of only 29 days. At the time of death, mice had marked splenomegaly along with leukemic cells circulating in the peripheral blood and infiltrating both the spleen and bone marrow. In contrast, mice injected with REH cells with HMGA1 silencing survived for >40 days (P<0.001) and had a significant decrease in tumor burden in the peripheral blood, spleen, and bone marrow. Similar results were obtained with 697 cells, although this model was more fulminant with control mice surviving for a median of only 17 days. To determine whether the leukemic blasts found in mice injected with ALL cells after HMGA1 silencing represented a clone that expanded because it escaped HMGA1 silencing, we assessed HMGA1 levels and found that cells capable of establishing leukemia had high HMGA1 expression, with levels similar to those observed in control cells without HMGA1 silencing. RNA-Seq analyses from REH and 697 cell lines with and without HMGA1 silencing revealed that HMGA1 up-regulates transcriptional networks involved in RAS/MAPK/ERK signaling while repressing the IDH1 metabolic gene, the latter of which functions in DNA and histone methylation. Studies are currently underway to identify effective agents to target HMGA1 pathways. Conclusions: Silencing HMGA1 dramatically disrupts leukemogenic phenotypes in vitro and prevents the development of leukemia in mice. Mechanistically, RNA-Seq analyses revealed that HMGA amplifies transcriptional networks involved cell cycle progression and epigenetic modifications. Our findings highlight the critical role for HMGA1 as a molecular switch required for leukemic transformation in pB-ALL and a rational therapeutic target that may be particularly relevant for relapsed B-ALL. Disclosures No relevant conflicts of interest to declare.

Separate Roles of Asparagine and Glutamine in Cytostatic Effect of L-Asparaginase - Stable Isotope Tracing Approach

L-asparaginase (ASNase) is one of the crucial components of acute lymphoblastic leukemia (ALL) therapy. Although we have previously shown that ASNase triggers metabolic reprogramming of leukemic cells, the significance and interconnections of the changes have not yet been elucidated. Metabolic reprogramming is an accompanying feature in therapy response and is also triggered by commonly used cytostatic drugs. ASNase hydrolyzes two non-essential amino acids asparagine (Asn) and glutamine (Gln). Therapeutic ASNase concentration used in vitro transforms all Asn and Gln to aspartate (Asp) and glutamate (Glu), respectively. We employed stable isotope tracing to understand the complexity and interconnection of metabolic processes altered in leukemia cells exposed to ASNase. Stable isotope tracing is a unique method enabling the quantification of metabolic flux. When substrate with 13C is metabolized by cells, enzymatic reactions rearrange 13C atoms resulting in specific labeling patterns in downstream metabolites. Therefore, this method allows us to determine exact pathways which are used to metabolize labeled nutrient. Glucose and Gln media concentrations were adjusted to 8mM and 1mM, respectively, to more resemble physiological conditions. All experiments were done with 0.8 IU/ml ASNase which is close to the plasma ASNase level in the induction phase of ALL treatment. We studied the effect of ASNase on two B-ALL cell lines with different sensitivity to this cytostatic drug: REH (IC50=0.000231 IU/ml) and NALM6 (IC50=0.328894 IU/ml). U13C glucose tracing revealed that although pyruvate and lactate intracellular concentrations are not changed after ASNase treatment, the relative flux from glucose to these metabolites (expressed as M+3 which represents 3 13C atoms in 1 molecule) is lowered (e.g. lactate (M+3): NALM6 - (0.5898±0.0026) vs (0.4473±0.0031), p<0.0001); REH - (0.3417±0.0023) vs (0.2411±0.0024), p<0.0001). Unlike glycolytic intermediates, the levels of tricarboxylic acid (TCA) cycle intermediates are lowered after ASNase administration (e.g. relative quantification of malate: NALM6 - (1.6120±0.1202) vs (0.4358±0.0247), p<0.0001; REH - (0.8231±0.0689) vs (0.2484±0.0573), p<0.0001). Interestingly, the relative flux from glucose to TCA cycle (expressed as e.g. malate (M+1-M+4) which represents at least 1 13C atom in a malate molecule) is increased in NALM6 ((0.0947±0.0021) vs (0.3605±0.0070), p<0.0001) and lowered in REH cells ((0.3109±0.0100) vs (0.1449±0.0185), p<0.0001) after ASNase. Since in our setting ASNase depletes both Asn and Gln, we tested what are the separate roles of Asn and Gln depletion in ASNase treatment. We discovered that omitting Gln from the media caused that TCA cycle intermediates are lowered in both cell lines to the same extent as after ASNase. Moreover, the flux from glucose to TCA cycle resembles the one after ASNase in both cell lines. In contrast the results of Asn withdrawal showed that level of TCA cycle intermediates are unchanged. The relative flux from glucose to TCA cycle is mildly downregulated without Asn in the media in both cell lines (e.g. malate (M+1-M+4): NALM6 - (0.1485±0.0014) vs (0.1158±0.0004), p<0.0001; REH - (0.2662±0.0031) vs (0.2024±0.0015), p<0.0001). Many studies emerged that highlight TCA cycle importance in Asp production and hence in sustaining cell viability and proliferation. Although TCA cycle is diminished after ASNase treatment, our results showed that both NALM6 and REH cells are able to maintain Asp and Glu levels. Using U13C Asp and U13C Glu, we discovered that, unexpectedly, both cell lines are able to import Asp and Glu from the media in a dose-dependent manner. Altogether, our results demonstrate different consequences of Asn and Gln depletion during ASNase treatment. According previous studies it is probably only Asn that is completely depleted after ASNase administration in ALL therapy with unchanged or lowered Gln concentration. Therefore, this fact should be considered in in vitro studies where Gln depletion could blur the effect of Asn depletion. Above that, we showed that leukemia cells are able to uptake Asp and Glu. This indicates the possible way how leukemic cells could cope with ASNase treatment. This work is supported by NV18-07-00129 and Charles University Grant agency 79421. Disclosures No relevant conflicts of interest to declare.

Chromatin Architecture Modulation in B-Cell Acute Lymphoblastic Leukemia Carrying DUX4 Fusions

B-cell acute lymphoblastic leukemia (B-ALL) carrying DUX4 fusions is a novel cluster of B-ALL. DUX4 fusions are generated from insertions of wild- type (WT) DUX4, mainly into the IGH locus.The translocation replaces the 3′ end of the WT DUX4 coding region with a fragment of IGH or another gene, producing DUX4 out-of-frame fusion proteins devoid of the C terminus of WT DUX4. Usually, WT DUX4 is expressed in germ cells in testis, while its expression is epigenetically repressed in somatic tissues. Recently, it is identified to plays a critical role in transcriptional programs at the cleavage of human fertilized egg. In B-ALL, DUX4-IGH (D-I) is shown to be essential for leukemic transformation; however, little is known about the mechanistic basis. Here in this study, we extensively investigated the biological effects of D-I. First, we assessed the role of D-I using in vitro cell culture assays with human cord blood (CB) CD34+ cells. Introduction of D-I significantly caused retention of the CD34+ cell population compared with the mock vector, even though it failed to preferentially promote differentiation toward B cell lineage in vitro. To analyze the epigenetic and transcription control by D-I, we performed chromatin immunoprecipitation coupled with sequencing (ChIP-seq) using cell lines. In NALM6, a B-ALL cell line carrying D-I, a subset of D-I binding sites is accompanied by H3K4me3 and H3K27ac. We also assessed the histone modification status in Reh cells, a B-ALL cell line without DUX4 fusions, and observed that active histone marks are detected after binding of ectopically expressed D-I. Nevertheless, RNA sequencing of NALM6 and Reh overexpressing D-I showed minimal activation of genes near the D-I binding sites compared with those of NALM6 overexpressing WT DUX4. WT DUX4 is known to preferentially bind and activate repeat elements, especially human endogenous retroviral (HERV) elements in embryonic cells. NALM6 cells overexpressing WT DUX4 showed a drastic increase in the expression of HERV elements, while NALM6 and Reh overexpressing D-I did not. The expression of HERV elements was not altered by D-I in all the genomic regions, and we did not observe increased expression of HERV elements in patient leukemia samples with DUX4 fusions as well. Furthermore, Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) showed that chromatin status was not affected by the binding of D-I at the D-I bound HERV element, indicating that transcriptional and insulating ability of WT DUX4 in these areas are lost in D-I. Next, we performed ATAC-seq using NALM6 cells, comparing the status between pre- and post- D-I knockdown. Genomic areas with decreased ATAC signal after knockdown of D-I are enriched in D-I binding sites, and ATAC signal was increased when we compared the status between pre- and post- induction of D-I in Reh cells. Through the immunoprecipitation of endogenous D-I in NALM6 cells, we identified SWI/SNF complex elements as binding partners of D-I, further highlighting the chromatin opening ability of D-I. Motif analysis of the genomic areas with decreased ATAC signal after knockdown of D-I identified only DUX4 motif as a significant motif, suggesting that D-I is not apparently cooperating with other transcription factors. On the other hand, ATAC signal was increased in substantial genomic areas after knockdown of D-I, and motif analysis identified SPI1, TCF3, and EBF1 motifs. Integrated analysis of transcriptome data also supports the idea that transcription factors related to B cell differentiation are repressed in the presence of D-I, and derepressed after knock down of D-I. Despite the attenuated transcriptional activity, B-ALL carrying DUX4 fusions manifests a characteristic expression pattern. D-I binding sites are not always relevant to the gene areas with increased transcriptions. Therefore, we compared the genomic areas where ATAC signal is raised by D-I, and genes whose expression is affected by D-I. We identified genes with ATAC signal change both in NALM6 cells with D-I knockdown and in Reh cells with D-I induction. We identified D-I binding in some of these genes, and the pharmacological inhibition of one of the genes caused cell death in NALM6 cells in vitro and in vivo, suggesting that this gene is the genuine target of D-I. In summary, our study elucidated the detailed difference of function between WT DUX4 and DUX4-IGH, and demonstrated the ability of DUX4-IGH as a chromatin modulator. Disclosures No relevant conflicts of interest to declare.

Anti-Apoptotic BCL-2 Family Members Confer Resistance to Calicheamicin-Based Antibody-Drug Conjugate Therapy of Acute Leukemia

BACKGROUND: With gemtuzumab ozogamicin (GO; targeting CD33) and inotuzumab ozogamicin (IO; targeting CD22), 2 antibody-drug conjugates delivering a toxic calicheamicin (CLM) derivative have recently been approved for the treatment of people with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), respectively. While effective in some, many patients do not benefit from these ADCs. It is unclear to what degree anti-apoptotic BCL-2 family members are involved in modulating efficacy of CLM-based ADCs, with limited studies coming to differing conclusions. Given the clinical availability of small molecule inhibitors for BCL-2 family proteins (BCLi), here we clarify the impact of BCL-2 family proteins on the anti-leukemic activity of CLM-ADCs. MATERIALS AND METHODS: Human AML and ALL cell lines were engineered to overexpress BCL-2, BCL-XL, and MCL-1 via lentiviral gene transfer. AML and ALL cell lines as well as AML patient samples were exposed to increasing concentrations of GO or IO with or without the BCL-2 inhibitor ABT-199 (venetoclax), the BCL-2/BCL-XL inhibitor ABT-263 (navitoclax), and the MCL-1 inhibitor AZD5991. Dead cells were enumerated by flow cytometry via 4',6-diamidino-2-phenylindole staining after 72 hours. For BH3 profiling of AML patient specimens, thawed AML patient specimen aliquots were exposed to JC-1 mitochondrial dye and BH3 peptides, and peptide-induced depolarization was then calculated as a percent relative to a CCCP positive control, yielding a priming score for each BH3 peptide. RESULTS: At a dose of 1000 pg/ml, GO killing of ML-1 (AML) cells decreased from 56±5% (mean±SEM) in parental cells to 32±7% (p<0.01) and 26±6% (p<0.01) in cells overexpressing BCL-2 and BCL-XL, respectively (all n=3). Similar results were seen in another AML cell line (HL-60). In REH (ALL) cells treated with IO, overexpression of BCL family members also reduced killing - at 500 pg/ml, 59±8% of cells were killed in contrast to 12±1% (p<0.01) of BCL-2-expressing and 11±1% (p<0.01) of BCL-XL-expressing cells, with similar results seen in another ALL cell line (RS4;11). Addition of ABT-199 or ABT-263 at 1 µM modestly increased GO-mediated killing of AML cell lines - for example, ML-1 cells treated with GO at 100 pg/ml, cytotoxicity increased from 41±6% to 57±7% (ABT-199, p<0.01) and 61±8% (ABT-263, p<0.01). The effect of BCLi was more pronounced on IO-mediated killing of ALL cell lines than on GO-mediated killing of AML lines. For example, killing of REH cells treated with IO at 25 pg/ml increased from 39±7% (without BCLi) to 72±8% (ABT-199 1 µM, p<0.01) and 87±9% (ABT-263 1 µM, p<0.01), with similar results seen in RS4;11 cells. BH3 peptide profiling of AML patient specimens treated with GO implicated MCL-1 as a potential additional modulator of AML response to GO. Consistent with this finding, overexpression of MCL-1 reduced leukemia cell death in HL-60 cells treated with GO (GO at 1000 pg/ml, 41±2 % vs. 26±1 %, p=0.01) and RS4;11 cells treated with IO (IO at 100 pg/ml, 76±2% vs. 27±6%, p<0.01). The MCL-1 inhibitor AZD5991 modestly increased the anti-leukemic efficacy of GO in ML-1 cells and AML patient specimens, but more dramatically enhanced IO killing of REH cells (IO at 10 pg/ml, 18±2% without AZD5991 vs. 70±2% with 0.1 µM AZD5991, p<0.01). The triplet combination of GO, ABT-199 and AZD5991 did not improve markedly on the ABT-199/AZD5991 combination in the absence of GO in cell lines or AML patient specimens, though the triplet combination of IO, ABT-199 and AZD5991 showed promising activity: in REH cells treated with 10 pg/ml IO, cytotoxicity was 18±2% without BCLi, 32±8% with ABT-199 0.1 µM, 19±2% with AZD5991 0.01 µM, and 56±14% with the triplet combination (p<0.01 for comparison of triplet combination with IO/BCLi doublet). CONCLUSIONS: Our studies establish an important role of anti-apoptotic BCL-2 family members as resistance factor for CLM-based ADC therapy of acute leukemia. These findings provide the rationale to explore the combination of small-molecule inhibitors of BCL-2 family members with CLM-ADCs as a combination strategy in the clinic to improve the efficacy of GO and, particularly, IO. These therapeutic strategies may incorporate the assessment of the relative contribution of specific BCL-2 family members to an individual cancer patient's disease. Disclosures Jean: Eutropics Pharmaceuticals: Employment. Cardone:Eutropics Pharmaceuticals: Employment, Equity Ownership. Walter:Seattle Genetics: Research Funding; Kite Pharma: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; New Link Genetics: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy.