The Combination Of Hypomethylating Agents and Histone Deacetylase Inhibitors(HDACi) Are Synergistically Cytotoxic and Reverse The Malignant Phenotype In Preclinical Models Of T-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 646-646 ◽  
Author(s):  
Owen A. O'Connor ◽  
Enrica Marchi ◽  
Kelly Zullo ◽  
Luigi Scotto ◽  
Jennifer E. Amengual ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Single Agent ◽  
Hematologic Malignancies ◽  
Advisory Committees ◽  
Methylation Array ◽  
Simultaneous Exposure ◽  
The Mean

Abstract Both HDAC inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) are known to influence global expression patterns in hematologic malignancies. Little is known about the combination of these two drug classes in lymphoid malignancies. HDACIs have marked single agent activity in the T- cell lymphomas (TCL), although the mechanism of action is not well defined. DNMTIs affect cytosine methylation of genomic DNA and have activity mainly restricted to the myeloid derived hematologic malignancies. The single agent efficacy and synergistic interaction of a panel of HDACIs (panobinostat, belinostat, romidepsin and vorinostat) and DNMTIs (decitabine (DEC), 5-azacytadine (5-AZA)) was evaluated in models of TCL. The molecular basis for the synergistic effect of HDACIs and DNMTIs was evaluated by gene expression profiling (GEP) and CpG methylation CTCL. Single agent concentration and time effect relationships were generated for 2 CTCL (HH, H9) and 2 T-ALL (P12, PF382) cell lines. Romidepsin and belinostat were the most potent HDACIs with the mean 48 hour IC50 of 8.8 nM (range 1.7-2.7 nM) and 85 nM (range 36-136 nM), respectively. Cell viability was not affected by treatment with DEC or 5-AZA at 24 and 48 hours at concentrations as high as 20 μM. Reduction in viability was first demonstrated after 72 hours of exposure to DEC, with the mean IC50 of 14.8 μM (range 0.4 μM- >20uM). Simultaneous exposure of combinations of DEC plus romidepsin or DEC plus belinostat at their IC10, IC20, and IC50 produced marked synergy in all TCL derived cell lines. Simultaneous exposure of DEC plus romidepsin demonstrated the deepest synergy at 72 hours with synergy coefficients in the range of 0.3. Cells treated with the combination of DEC plus romidepsin also demonstrated significant induction of apoptosis as evaluated by annexinV/propridium iodide via FACS analysis and an increase in acetylated histone 3 by immunoblot. The in vivo activity of the combination of DEC plus belinostat was investigated in a xenograft model of CTCL using HH, the most resistant TCL derived cell line. Mice were treated with DEC 1.5 mg/kg (day 29, 33, 35, 37, 39, 41, 43) and/or belinostat 100 mg/kg (day 29-day 47). The combination mouse cohort demonstrated statistically significant tumor growth delay compared to DEC alone (p=0.002) and belinostat alone (p=0.001). The interaction of DEC and romidepsin was analyzed by GEP and methylation array. Interestingly, the baseline malignant phenotype seen in the CTCL cell-lines was reversed. A significant down-regulation of genes involved in biosynthetic pathways including protein and lipid synthesis, and a significant up-regulation of genes responsible for cell cycle arrest were seen. The vast majority (114/138; 92%) of genes modulated by the single agents were similarly modulated by the combination. However, the latter induced a further significant change in the transcriptome, affecting an additional 390 genes. Similarly, methylation array data was analyzed following treatment of these drugs alone and in combination. DEC induced de-methylation of 190 different gene regions corresponding to 175 genes and an additional 335 loci. Interestingly, when combined with romidepsin the number of demethylated gene regions decreased to 85 corresponding to 79 genes, 78 of which were common with DEC and 148 additional loci. The comparison of gene expression and methylation demonstrated a significant inverse relationship (R2 = 0.657) with genes found to be differentially expressed in GEP and methylation analysis. (Figure 1)Figure 1Summary of gene expression and methylation analysis.Figure 1. Summary of gene expression and methylation analysis. These data support the observation that DNMTIs in combination with HDACIs produces significant synergistic activity in models of TCL. Further evaluation of the mechanism of action with DNMTIs in combination with HDACIs is ongoing, and a clinical trial of the combination is now open. Disclosures: O'Connor: Celgene Pharmaceuticals: Consultancy; Spectrum Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Allos Therapeutics: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Off Label Use: Hypomethylating Agents in T-cell lymphoma. Amengual:Acetylon Pharmacueticals, INC: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Epigenetic Targeting with EZH2 and HDAC Inhibitors Is Synergistic in EZH2 Deregulated Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 839-839 ◽  
Author(s):  
Jennifer Kimberly Lue ◽  
Sathyen A Prabhu ◽  
Yuxuan Liu ◽  
Owen A. O'Connor ◽  
Jennifer E Amengual
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Lymphoma Cell ◽  
Advisory Committees ◽  
Genetics Research ◽  
Transcriptional Inhibition

Abstract EZH2 is critical in a process known as the Germinal Center (GC) reaction during which B-cells undergo somatic hypermutation and isotype switching in order to develop a large antibody repertoire. EZH2 is a histone methyltransferase serving as the catalytic subunit of the Polycomb Repression Complex 2 (PRC2), which is responsible for tri-methylation of histone 3 lysine 27 (H3K27), a mark of transcriptional repression. EZH2 recruits HDAC1/2 and DNMTs through its cofactor EED to further inhibit transcription. Mutations in EZH2 are found in 7-12% of FL and 22% of GC-DLBCL. EZH2 overexpression secondary to MYC and miRNA dysfunction has also been described. EZH2 also plays a role in T-cell differentiation and has been found in various T-cell malignancies. Histone acetyltransferases (HAT), notably CBP and p300, have also been implicated in B- and T-cell lymphomas and are mutated/deleted in 39% of GC-DLBCL and 41% of FL. Given the presence of EZH2 and HAT dysregulation in lymphoma, we evaluated the potential synergy of EZH2 and HDAC inhibitors co-treatment. Single agent activity for GSK126, an EZH2 inhibitor, and romidepsin, a pan-HDAC inhibitor, was established in a panel of lymphoma cell lines (GC-DLBCL, Non-GC DLBCL, MCL and T-Cell lymphoma, n=21). Cell lines with known EZH2 dysregulation (GC-DLBCL and ATLL) were more sensitive to EZH2 inhibitors as exhibited by lower half maximal effective concentration (EC50) after 6 day exposure (EC50 0.01-16 µM). There was no association between HAT mutation/deletion and romidepsin sensitivity. A panel of lymphoma cell lines was treated for 72 hr with GSK126 and romidepsin using concentrations represented by their EC30-50 (0.5-4.0 µM), and EC20-40 (1.0-4.0 nM), respectively. Synergy was assessed by Excess over Bliss (EOB), where EOB > 10 represents synergy. Simultaneous exposure to GSK126 and romidepsin in GC-DLBCL cell lines demonstrated potent synergy as represented by EOB > 30. Synergy was also present in ATLL cell lines (EOB 28), which are known to have EZH2 dysregulation, as well as non-GC DLBCL cell lines (EOB 47). Although these cell lines do not have EZH2 mutations, some possess relative EZH2 over-expression compared to other lymphomas. Evaluation of drug schedule using GSK126 pretreatment prior to romidepsin exposure did not impact synergy. Compared to single agent activity, the combination of GSK126 (2 µM) and romidepsin (1-4 nM) led to a more pronounced decrease in H3K27 tri-, di-, and mono-methylation and increased acetylation in 4 GC-DLBCL cell lines (OCI-LY7, Pfeiffer, SU-DHL-6, SU-DHL-10) at 24 or 48 hrs. The impact of the combination on the function of the PRC2 complex was assessed via co-immunoprecipation in these cell lines. The combination demonstrated dissociation of the PRC2 complex (EZH2, SUZ12, EED, and RbAp46/48) as compared to single agent exposure. Treatment with the combination also induced dissociation of HDAC2 and DNMT3L. In addition, we observed decreased protein expression of PRC2 complex members and increased p21/CDKN1A, which was more notable in the combination treatment as compared to single agent. This may be due to the removal of HDACs from the p21 transcriptional start site through the disruption of the PRC2 complex and direct inhibition of HDACs, thus leading to increase expression of p21. The combination also led to decreased nuclear localization of EZH2 and its cofactors. Apoptosis was confirmed by caspase 3 and PARP cleavage, and was more potently cleaved after exposure to the combination. Based on the findingthat HDAC2 dissociated from PRC2 complex after treatment with GSK126 and romidepsin, a selective HDAC1/2 inhibitor, ACY-957 (Acetylon Pharmaceuticals), was combined with GSK126 which demonstrated potent synergy in 4 GC-DLBCL cell lines (EOB 37). This data suggests that concomitant inhibition of EZH2 and HDAC is highly synergistic and leads to the dissociation of PRC2 complex. By releasing transcriptional inhibition key tumor suppressors and cell cycle regulators may be re-expressed. Potency of this epigenetic combination may be predicted by gene expression signatures for which RNA-seq libraries are currently in production. Reversing transcriptional inhibition using a combination of EZH2 inhibitors and HDAC inhibitors may lead to a potent treatment option for lymphomas dependent upon EZH2 and HAT activity. Figure 1 Figure 1. Disclosures O'Connor: Seattle Genetics: Research Funding; Spectrum: Research Funding; Seattle Genetics: Research Funding; Spectrum: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Celgene: Research Funding. Amengual:Acetylon Pharmaceuticals: Research Funding; Bristol-Myers Squibb: Research Funding.

The Aurora A Kinase Inhibitor, Alisertib, Has Broad Activity In Nonclinical Models Of T-Cell Lymphoma and Is Highly Synergistic With Romidepsin, But Not With Pralatrexate Or The Proteasome Inhibitor, Ixazomib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5141-5141 ◽  
Author(s):  
Kelly Zullo ◽  
Luigi Scotto ◽  
Jennifer E. Amengual ◽  
Owen A. O'Connor
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Time Effect ◽  
T Cell Lymphoma ◽  
Aurora A Kinase ◽  
Advisory Committees

Abstract Aurora A kinase (AAK), a serine-threonine protein kinase, regulates mitotic entry, spindle formation, and cytokinesis. Alisertib is a selective AAK investigational inhibitor with demonstrated clinical activity in acute myeloid leukemia, peripheral t-cell lymphoma (PTCL), DLBCL and other heme-lymphatic cancers. Here we report the cytotoxicity and apoptotic effect of Alisertib  in a panel of T-cell-derived lymphoma cell-lines (TCL) (CTCL, HTLV+, T-ALL) and B-cell lymphoma cell-lines (DLBCL-ABC, DLBCL-GCB, MCL) alone and in combination with romidepsin, pralatrexate (PDX) and ixazomib, a  proteasome inhibitor. Single agent concentration and time effect relationships were generated for 8 TCL, 4 DLBCL (2 ABC, 2 GCB) and 4 MCL cell-lines. The mean IC50 of alisertib in TCL was 350 nM (range 100-1000nM) and in B-cell lymphoma lines (DLBCL, MCL) was 200 nM (range 20-300 nM) at 48 hours, measured by growth inhibition. In all cell lines evaluated, there was a consistent 2-log fold decrease in IC50 values at 72 hours.  Combination studies evaluating synergy were performed testing schedule, concentration, and time effect relationships. Interestingly, simultaneous exposure of combined alisertib and romidepsin at their IC10, IC20, and IC30 demonstrated marked synergy in TCL. Deepest synergy was observed at 72 hours with synergy coefficients ranging from 0.2 to 0.7. This synergistic interaction was restricted to the TCL cell-lines, with no benefit demonstrated in DLBCL or MCL cell lines. (Table 1) Similarly, alisertib did not demonstrate synergy in TCL, DLBCL or MCL cell lines at any concentration, combination, or time schedule  with PDX  or ixazomib (simultaneous incubation of alisertib + PDX, 24 hour pre-exposure alisertib followed by PDX, 24 hour pre-exposure PDX followed by alisertib; simultaneous incubation of alisertib + ixazomib).Table 1Synergy Coefficients of Alisertib in Combination with Romidepsin at 72 Hours.Combination DND41(T-ALL) J.CAM1.6(T-ALL) HH(CTCL) H9(CTCL) C5MJ(HTLV+) Romidepsin [IC10-20] +Alisertib 50 nM 0.96 0.81 1.05 1.1 1.53 Romidepsin +Alisertib 100nM 0.51 0.56 0.68 0.66 0.58 Romidepsin +  Alisertib 1000nM 0.40 0.20 0.40 0.46 0.70 1 1=additive; <1=synergistic; >1= subadditive Evidence for apoptosis was confirmed for alisertib in combination with romidepsin in the TCL cell-line, H9 after 48 hours of exposure by increased caspase 3 and PARP cleavage,  acetylated H3 expression, and decreased Cyclin B1, P27, and BCL2 expression; suggesting that cell death occurred through apoptosis.  AnnexinV/propridium iodide via FACS analysis confirmed induction of apoptosis. These data support the observation that alisertib produces broad single-agent activity in models of TCL and demonstrates marked synergy with romidepsin. Interestingly this effect is restricted to TCL and there is no synergistic effect with ixazomib or PDX in TCL.  Further evaluation of the mechanism of action with alisertib in combination with romidepsin as well as in vivo modeling of these combinations is ongoing. Disclosures: Off Label Use: Aurora kinase inhibitors are not approved for T-Cell lymphoma. Amengual:Acetylon Pharmaceuticals, INC: Membership on an entity’s Board of Directors or advisory committees, Research Funding. O'Connor:Millennium Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Spectrum Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Allos Therapeutics: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Celgene Pharmaceuticals: Consultancy.

Low Expression Of The FUCA1 Gene Is An Adverse Prognostic Factor In Myeloma and Combined With High Sialyltransferase Gene Expression Identifies Patients At Increased Risk Of Early Disease Progression and Death

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1864-1864 ◽  
Author(s):  
Siobhan Glavey ◽  
Ping Wu ◽  
Laura S Murillo ◽  
Catherine Loughrey ◽  
Aldo M Roccaro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Prognostic Significance ◽  
Advisory Committees ◽  
Methylation Array ◽  
Selectin Ligands ◽  
Mapping Array ◽  
Low Expression

Abstract Background Glycosylation is a post translational modification which results in the addition of carbohydrate determinants to proteins and lipids. This has a major influence on the function of molecules known to be important in myeloma cell adhesion and trafficking such as integrins and selectins. We previously reported that ST3GAL6, a sialyltransferase involved in the synthesis of functional selectin ligands in humans, is over expressed in myeloma cells and associated with inferior survival in the MRC Myeloma IX study (MRC IX). We now extend our analysis of the MRC IX dataset to evaluate the potential prognostic significance of other glycosylation genes that are differentially regulated between normal and malignant plasma cells. Methods Analyzing publically available microarray transcriptomic datasets (Mayo Clinic GSE6477, University of Arkansas (UAMS) GSE24080, GSE2658) we first identified dysregulated glycosylation genes in MM. The prognostic significance of these candidate genes in MRCIX (singly and in combination) was analyzed using Kaplan Meier survival estimates for progression free survival and overall survival (PFS, OS). These results were validated in independent datasets (TT2 and TT3). Any genes significantly associated with survival were correlated with FISH abnormalities and ISS and multivariate analysis was performed to determine their independence as prognostic factors. QPCR was performed in cell lines to validate GEP findings. Matching SNP-based mapping array and methylation array data were analyzed. Membrane protein extracts from MM cell lines were applied directly to lectin microarrays following fluorescent labeling to generate cell surface glycan profiles. Immunohistochemistry for glycosylation enzymes and glycans was performed on patient bone marrow samples. Results High expression (top quartile as cut-off) of the sialyltransferase gene ST3GAL1 showed a trend towards inferior OS (median survival 35 mos .v. 45 mos, p=0.07) with significantly reduced PFS (19 .v. 14 mos, p=0.015). Increased expression of ST3GAL1 correlated with the presence of t(4;14) (p=0.009), del13q (p=0.001), +1q (p=0.01) and hypodiploidy (p=0.00001). Low expression (lower quartile by GEP) of the gene FUCA1, which encodes tissue alpha-L-fucosidase, was linked to inferior outcome (median OS 44 .v. 38 mos, p=0.025). On multivariate analysis low FUCA1 expression was independent of other important prognostic factors (HR=1.61, p=0.017). Patients with t(11;14) by FISH were more likely to have low FUCA1 than t(11;14) negative patients. There was no significant association between ISS and ST3GAL6, ST3GAL1 or FUCA1. We verified the effect of low FUCA1 in the GSE24080 dataset from TT2 and TT3 combined. Patients with low FUCA1 had significantly worse OS (p=0.0002). SNP mapping array and methylation array analysis did not show any evidence of correlation between copy number alterations or hypermethylation of FUCA1 and its low expression level. Since FUCA1 participates in N-glycan degradation with removal of fucose residues, this raises the possibility that a reduction in FUCA1 may lead to excessive fucosylation of cancer related glycans involved in adhesion and trafficking, such as sLex. Indeed, lectin arrays of MM cell lines revealed high levels of a-1,3/a-1,6 linked fucose, as determined by binding of Aleuria Aurantia Lectin (AAL). The HECA-452 antibody recognizes a functional trisaccharide domain shared by sialyl Lewis a and sLex and known to bind to E-selectin. Preliminary immunohistochemistry revealed increased HECA452 staining in bone marrow samples, which had both strong ST3GAL6 and low FUCA1 staining. Combining gene expression data showed that reduced expression of FUCA1 with increased expression of either ST3GAL6 or ST3GAL1 or both, identified a subgroup of patients (18%) in MRC Myeloma IX with particularly poor outcome (figure 1). Similar results were found in TT2 and TT3 with 17% of patients affected. Conclusions Altered glycosylation gene expression patterns may identify patients at high risk of disease progression and early death. Our data implicates sialyltransferases and selectin ligands as potential therapeutic targets in MM. Disclosures: Morgan: Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millenium: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Johnson and Johnson: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

scholarly journals Euroclonality-NGS DNA Capture Panel for Integrated Analysis of IG/TR Rearrangements, Translocations, Copy Number and Sequence Variation in Lymphoproliferative Disorders

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 888-888 ◽  
Author(s):  
Peter Stewart ◽  
Jana Gazdova ◽  
Nikos Darzentas ◽  
Dorte Wren ◽  
Paula Proszek ◽  
...  
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Committees ◽  
Proficiency Assessment ◽  
Dna Capture ◽  
Tumour Infiltration ◽  
T Cell Malignancies ◽  
Cell Clonality

Introduction: Current diagnostic standards for lymphoproliferative disorders include detection of clonal immunoglobulin (IG) and/or T cell receptor (TR) rearrangements, translocations, copy number alterations (CNA) and somatic mutations. These analyses frequently require a series of separate tests such as clonality PCR, fluorescence in situ hybridisation and/or immunohistochemistry, MLPA or SNParrays and sequencing. The EuroClonality-NGS DNA capture (EuroClonality-NDC) panel, developed by the EuroClonality-NGS Working Group, was designed to characterise all these alterations by capturing variable, diversity and joining IG and TR genes along with additional clinically relevant genes for CNA and mutation analysis. Methods: Well characterised B and T cell lines (n=14) representing a diverse repertoire of IG/TR rearrangements were used as a proficiency assessment to ensure 7 testing EuroClonality centres achieved optimal sequencing performance using the EuroClonality-NDC optimised and standardised protocol. A set of 56 IG/TR rearrangements across the 14 cell lines were compiled based on detection by Sanger, amplicon-NGS and capture-NGS sequencing technologies. For clinical validation of the NGS panel, clinical samples representing both B and T cell malignancies (n=280), with ≥ 5% tumour infiltration were collected from 10 European laboratories, with 88 (31%) being formalin fixed paraffin-embedded samples. Samples were distributed to the 7 centres for library preparation, hybridisation with the EuroClonality-NDC panel and sequencing on a NextSeq 500, using the EuroClonality-NDC standard protocol. Sequencing data were analysed using a customised version of ARResT/Interrogate, with independent review of the results by 2 centres. All cases exhibiting discordance between the benchmark and capture NGS results were submitted to an internal review committee comprising members of all participating centres. Results: All 7 testing centres detected all 56 rearrangements of the proficiency assessment and continued through to the validation phase. A total of 10/280 (3.5%) samples were removed from the validation analysis due to NGS failures (n=1), tumour infiltration &lt; 5% (n=7), and sample misidentification (n=2). The EuroClonality-NDC panel detected B cell clonality (i.e. detection of at least one clonal rearrangement at IGH, IGK or IGL loci) in 189/197 (96%) B cell malignancies. Seven of the 8 discordant cases were post-germinal centre malignancies exhibiting Ig somatic hypermutation. The EuroClonality-NDC panel detected T cell clonality (i.e. detection of at least one clonal rearrangement at TRA, TRB, TRD or TRG loci) in 70/73 (96%) T cell malignancies. In all 3 discordant cases analysis of benchmark PCR data was not able to detect clonality at any TR loci. Next, we examined whether the EuroClonality-NDC panel could detect clonality at each of the individual loci, resulting in sensitivity values of 95% or higher for all IG/TR loci, with the exception of those where limited benchmark data were available, i.e. IGL (n=3) and TRA (n=7). The specificity of the panel was assessed on benign reactive lesions (n=21) that did not contain clonal IG/TR rearrangements based on BIOMED-2/EuroClonality PCR results; no clonality was observed by EuroClonality-NDC in any of the 21 cases. Limit of detection (LOD) assessment to detect IG/TR rearrangements was performed using cell line blends with each of the 7 centres receiving blended cell lines diluted to 10%, 5.0%, 2.5% and 1.25%. Across all 7 centres the overall detection rate was 100%, 94.1%, 76.5% and 32.4% respectively, giving an overall LOD of 5%. Sufficient data were available in 239 samples for the analysis of translocations. The correct translocation was detected in 137 out of 145 cases, resulting in a sensitivity of 95%. Table 1 shows how translocations identified by the EuroClonality-NDC protocol were restricted to disease subtypes known to harbour those types of translocations. Analysis of CNA and somatic mutations in all samples is underway and will be presented at the meeting. Conclusions: The EuroClonality-NDC panel, with an optimised laboratory protocol and bioinformatics pipeline, detects IG and TR rearrangements and translocations with high sensitivity and specificity with a LOD ≤ 5% and provides a single end-to-end workflow for the simultaneous detection of IG/TR rearrangements, translocations, CNA and sequence variants. Table. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Klapper:Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Ferrero:Gilead: Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Membership on an entity's Board of Directors or advisory committees; Servier: Speakers Bureau. van den Brand:Gilead: Speakers Bureau. Groenen:Gilead: Speakers Bureau. Brüggemann:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy. Langerak:Gilead: Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Research Funding; Genentech, Inc.: Research Funding; Janssen: Speakers Bureau. Gonzalez:Roche: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Upregulated JAK/STAT Signaling Represents a Major Mode of Resistance to HDAC Inhibition In Lymphoma and Provides a Rationale for Novel Combination Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 434-434 ◽  
Author(s):  
Jason Smith ◽  
Katherine J. Walsh ◽  
Cassandra L Jacobs ◽  
Qingquan Liu ◽  
Siyao Fan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Single Agent ◽  
Acquired Resistance ◽  
Hematologic Malignancies ◽  
Cross Resistance ◽  
Clinical Activity ◽  
Molecular Pathways ◽  
Major Mode ◽  
High Degree

Abstract Abstract 434 Background Histone deacetylase inhibitors (HDACis) have demonstrated significant clinical activity in hematologic malignancies; however, single agent response rates have ranged between 20–50% with the duration of response often measured in months, suggesting that drug resistance is a major mode of failure. The pathways through which these agents work and the means by which tumors develop resistance to them are poorly understood. Combination therapy targeting multiple oncogenic pathways holds the promise to improve upon both the depth and durability of these responses. We investigated the mechanisms of inherent and acquired resistance to HDACis in a broad range of lymphomas. By detailing the molecular pathways implicated in resistance to HDACi, we sought to identify novel combinations of compounds that could overcome potential mechanisms that confer resistance. Methods and Results We tested two separate HDACis, LBH589 and SAHA in 51 cell lines representing a wide range of lymphomas including Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma, and Hodgkin lymphoma. Gene expression array data was generated for all these cell lines. We then identified genes that were significantly associated with resistance to both LBH589 and SAHA (p<.01) and applied hierarchical clustering to identify the functional significance of these genes. Histology was not predictive of sensitivity to either HDACi. These data were then analyzed using gene set enrichment to identify known molecular pathways associated with resistance. Activation of JAK/STAT signaling was found to be a major determinant of resistance among the cell lines that were relatively resistant to HDACi. (P<0.001, FDR <.25). To determine whether these genes that we found to be associated with resistance reflected potential mechanisms of acquired resistance to HDACi therapy, we separately engineered resistance to LBH589 and SAHA in three DLBCL cell lines (LY3, BJAB, Farage) through incremental dose escalation over a period of up to 6 months. Each of these three cell lines demonstrated sustained growth at drug concentrations that were at or above their original IC50. Each of these cell lines were then exposed to the other HDACi and tested for cross resistance. In each case, the cell lines demonstrated complete cross-resistance to the other drug. We then profiled the gene expression of these cell lines that had acquired resistance. Similar to our previous results, these cell lines demonstrated increased signaling through the JAK/STAT pathway, suggesting that mechanisms of inherent and acquired resistance are similar. We therefore reasoned that combining HDAC and JAK inhibition may overcome both inherent and acquired resistance. To investigate this hypothesis, we tested LBH589 and INCB018424, a JAK1/2 inhibitor, alone and in combination in the LY3, TMD-8, U2932, and BJAB cell lines. While INCB018424 demonstrated no single agent cytotoxicity, it yielded a high degree of synergy when combined with LBH589 with the combination index computed by the Chou-Talalay method ranging from .19 to .9. Conclusion HDACis show single agent activity in the treatment of a number of hematologic malignancies, however most patients develop resistance to these drugs after relatively short-lived remissions. Thus, the greatest promise of these drugs may lie in combination with other agents that target molecular pathways that underlie resistance to these drugs. Using gene expression profiling of a broad range of tumor types and sensitivity to HDACis we were able to identify activation of the JAK/STAT pathway as a common feature of inherent and acquired resistance to HDACis. We combined the JAK1/2 inhibitor INCB018424 with LBH589 and demonstrated a high degree of synergy. As the number of small molecule inhibitors with clinical activity increases, the need to identify rational preclinical combinations becomes greater. Pairing gene expression profiling and resistant cell lines is a promising approach to the selection of combinations likely to maximize clinical benefit while limiting toxicity. Disclosures: No relevant conflicts of interest to declare.

Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13569-e13569
Author(s):  
Enrica Marchi ◽  
Matko Kalac ◽  
Danielle Bongero ◽  
Christine McIntosh ◽  
Laura K Fogli ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Molecular Level ◽  
Cell Lymphoma ◽  
Single Agent ◽  
T Cell Lymphoma ◽  
Cytotoxicity Assays

e13569 Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.

scholarly journals Tolinapant (ASTX660), a Non-Peptidomimetic Antagonist of cIAP1/2 and XIAP, and the HDAC Inhibitor Romidepsin Are Synergistic in in Vitro Models of T Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4356-4356
Author(s):  
John S Manavalan ◽  
Ipsita Pal ◽  
Aidan Pursley ◽  
George A. Ward ◽  
Tomoko Smyth ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Tnf Alpha ◽  
Sensitive Cell ◽  
Advisory Committees

Abstract Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). Figure 1 Figure 1. Disclosures Smyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

scholarly journals Targeting BTN2A1 By a Unique Activating Mab Improves Vγ9Vδ2 T Cell Cytotoxicity Against Primary Acute Lymphoblastic Blasts

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2302-2302
Author(s):  
Anne-Charlotte Le Floch ◽  
Caroline Imbert ◽  
Aude De Gassart ◽  
Florence Orlanducci ◽  
Aude Le Roy ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemic Cells ◽  
Target Cells ◽  
Advisory Committees ◽  
Vγ9vδ2 T Cells ◽  
Privately Held

Abstract Introduction Vγ9Vδ2 T cells are new promising cytotoxic effectors in hematological malignancies. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials. Acute lymphoblastic leukemia (ALL) includes very heterogeneous clinico-biological entities, for which recent immunotherapy approaches are currently being developed. Nevertheless, global prognosis of ALL patients still be poor with a 5 years-overall survival of less than 40% and therefore, treatments need to be improved. Very few data are currently available on susceptibility of ALL blasts to Vγ9Vδ2 T cell cytotoxic activity. Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A molecules are targeted at clinical level, with the ICT01 agonist monoclonal antibody (mAb), that is currently tested in a multicentric phase ½ study (EVICTION study). Biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 is mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown. Here, we show that allogenic and autologous Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts, and we report that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1. Material and methods 5 ALL cell lines (697, RS4;11, NALM-6, HPB-ALL, SUP-T1) and PBMC from 11 adults ALL patients at diagnosis (B-ALL, T-ALL and Ph+ ALL) were tested in functional assays. We evaluated apoptosis of ALL cell lines and of primary ALL blasts after coculture with allogenic Vγ9Vδ2 T cells. ALL samples were also tested for their expansion capacities and a degranulation assay was performed at D14. We assessed in parallel relative quantification of the level expression of BTN2A1 (ICT0302 and 7.48 epitopes), and BTN3A (20.1 and 108.5 epitopes) on surface of ALL blasts. DAUDI-BTN2AKO+2A1 and HEK293-BTN2AKO+2A1 cells were used in binding assays, and modulation of TCR binding was assessed using recombinant tetramerized Vγ9Vδ2 TCR. Results We showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against ALL cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 ICT0302 agonist mAb significantly enhanced Vγ9Vδ2 T cells mediated apoptosis in comparison to control condition, even for the less spontaneously susceptible cells. We confirmed these observations with degranulation of autologous Vγ9Vδ2 T cells expanded from 5 ALL patients at diagnosis that was increased after treatment with anti-BTN2A1 ICT0302 agonist mAb. BTN3A and BTN2A1 were detected on surface of ALL blasts, and BTN3A 108.5 was the most expressed epitope. Interestingly, we observed that anti-BTN2A1 ICT0302 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells using with HEK293 and DAUDI cells. Discussion Our results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as well as BTN2A1 mAbs. These data bring novel understanding on the biology of BTN2A1 on leukemic cells and our ability to enhance both binding and function. These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types. Disclosures De Gassart: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Cano: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Olive: Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Anti-BTN2A1 ICT0302 is a murine agonist monoclonal antibody targeting BTN2A1 whose aim is to increase Vgamma9Vdelta2 T cells functions.

scholarly journals Evaluating Hematologist's Knowledge of CAR T-Cell Therapy in Hematologic Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2269-2269
Author(s):  
Lauren Willis ◽  
Sara R. Fagerlie ◽  
Sattva S. Neelapu
Keyword(s):  
T Cell ◽  
Cell Therapy ◽  
Board Of Directors ◽  
Research Funding ◽  
Hematologic Malignancies ◽  
Advisory Committees ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Background: The objective of this study was to assess current clinical practices of hematologist/oncologist (hem/onc) specialists related to chimeric antigen receptor (CAR) T-cell therapy in hematologic malignancies, in order to identify knowledge, competency, and practice gaps and barriers to optimal care. Methods: A continuing medical education (CME)-certified clinical practice assessment consisting of 25 multiple choice questions was developed to measure knowledge, skills, attitudes, and competence of hem/onc specialists regarding CAR T-cell therapy. The survey instrument was made available online to physicians without monetary compensation or charge. Respondent confidentiality was maintained, and responses were de-identified and aggregated prior to analyses. The activity launched on December 22, 2017 with global distribution, and participant responses are still being collected at the time of abstract submission. Results: At the time of this report there are 192 hem/onc activity participants, collection is on-going. Demographics are listed in Table 1 and levels of confidence and barriers to incorporating CAR T-cell therapy are listed in Table 2.Foundational KnowledgeSub-optimal knowledge was demonstrated in the area of CAR components, dosing, and FDA-approved indications.Over half (61%) could not correctly identify the components of a CAR construct (antigen-specific domain and the signaling domain).Almost half (45%) of the participants did not recognize that currently approved CAR T-cell therapies are dosed as a single infusion.25% demonstrated inaccurate knowledge by recommending patients wait 4 weeks after CAR T-cell infusion before driving.Over half (62%) of participants could not identify the FDA-approved indication for axicabtagene ciloleucel.Knowledge of Clinical Trial DataVery low awareness of efficacy data seen with various CAR T-cell products used to treat R/R B-cell ALL (ELIANA trial), R/R DLBCL (ZUMA-1, JULIET, TRANSCEND trials).Only 32% identified the correct CR/CRi rate seen with tisagenlecleucel in the ELIANA trial.Only 25% correctly identified the CR rate seen with axicabtagene ciloleucel in the ZUMA-1 trial.Only 32% demonstrated knowledge of the 6-month DFS rate for patients in the JULIET trial that had a CR at 3 months.Only 25% identified the association between the dose of JCAR017 and response rates from the TRANSCEND trial.Knowledge and Competence Managing Adverse EventsLack of competence recognizing and treating CAR T-cell associated adverse events such as cytokine release syndrome (CRS) and neurotoxicity.Almost half (44%) could not identify signs of CRS associated with CAR T-cell therapy and 43% lack knowledge that elevated serum C-reactive protein (CRP) is associated with the highest level of CRS (in patients with lymphoma receiving axicabtagene ciloleucel).41% could not identify that the mechanism of tocilizumab is to block IL-6 signaling.Over a third (35%) were unable to identify signs/symptoms/causes of neurotoxicity associated with CAR T-cell therapy.More than half of the learners (54%) could not identify the appropriate role of corticosteroid therapy after CAR T-cell administration in managing CRS and neurotoxicity. Conclusions: This activity found knowledge and competence deficits for hem/onc practitioners related to using CAR T-cell therapy for the treatment of patients with hematologic malignancies. Additionally, the activity demonstrated large gaps in confidence discussing CAR T-cell therapy with patients/families and managing adverse events. There is sub-optimal awareness of CAR T-cell foundational knowledge, clinical trial data, and recognition of common therapy related adverse events and management strategies. Additional education is needed to improve the knowledge, competence, and confidence of academic and community hem/onc specialists who care for patients with hematologic malignancies receiving CAR T-cell therapy as well as strategies for integrating novel agents into clinical practice. Disclosures Neelapu: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Karus: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Targeting Wnt/β-Catenin and BCL-2, Two Critical Survival Factors, Synergistically Induces Apoptosis in AML Via Rac1-Mediated MCL-1 Inhibition

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1442-1442
Author(s):  
Xiangmeng Wang ◽  
Po Yee Mak ◽  
Wencai Ma ◽  
Xiaoping Su ◽  
Hong Mu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Critical Survival

Abstract Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P<0.001 vs control, C-82, and ABT-199) in 10 out of 11 samples (CI=0.394±0.063, n=10). This synergy was also shown when AML cells were co-cultured with MSCs (P<0.001 vs control, C-82, and ABT-199) in all 11 samples (CI=0.390±0.065, n=11). Importantly, the combination also synergistically killed CD34+ AML stem/progenitor cells cultured alone or co-cultured with MSCs. To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P<0.0001 for ABT-199 and the combination) and that the combination was more effective than each single agent (P<0.001 vs C-82 or ABT-199) at 2 weeks of therapy. The combination also significantly decreased the leukemia burden in mouse spleens compared with controls (P=0.0046) and single agent treated groups (P=0.032 or P=0.020 vs C-82 or ABT-199, respectively) at the end of the treatment. However, the combination did not prolong survival time, likely in part due to toxicity. Dose modifications are ongoing. These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

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