Santhana G. T. Devaraj

Highly Active Combination of BRD4 Antagonist and Histone Deacetylase Inhibitor against Human Acute Myelogenous Leukemia Cells

The bromodomain and extra-terminal (BET) protein family members, including BRD4, bind to acetylated lysines on histones and regulate the expression of important oncogenes, for example, c-MYC and BCL2. Here, we demonstrate the sensitizing effects of the histone hyperacetylation-inducing pan–histone deacetylase (HDAC) inhibitor panobinostat on human acute myelogenous leukemia (AML) blast progenitor cells (BPC) to the BET protein antagonist JQ1. Treatment with JQ1, but not its inactive enantiomer (R-JQ1), was highly lethal against AML BPCs expressing mutant NPM1c+ with or without coexpression of FLT3-ITD or AML expressing mixed lineage leukemia fusion oncoprotein. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of c-MYC and BCL2 and reduced their levels in the AML cells. Cotreatment with JQ1 and the HDAC inhibitor panobinostat synergistically induced apoptosis of the AML BPCs, but not of normal CD34+ hematopoietic progenitor cells. This was associated with greater attenuation of c-MYC and BCL2, while increasing p21, BIM, and cleaved PARP levels in the AML BPCs. Cotreatment with JQ1 and panobinostat significantly improved the survival of the NOD/SCID mice engrafted with OCI-AML3 or MOLM13 cells (P < 0.01). These findings highlight cotreatment with a BRD4 antagonist and an HDAC inhibitor as a potentially efficacious therapy of AML. Mol Cancer Ther; 13(5); 1142–54. ©2014 AACR.

BET Protein Antagonist JQ1 Is Synergistically Lethal with FLT3 Tyrosine Kinase Inhibitor (TKI) and Overcomes Resistance to FLT3-TKI in AML Cells Expressing FLT-ITD

Recently, treatment with bromodomain and extraterminal protein antagonist (BA) such as JQ1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia (AML) cells, including those expressing FLT3-ITD. Here, we demonstrate that cotreatment with JQ1 and the FLT3 tyrosine kinase inhibitor (TKI) ponatinib or AC220 synergistically induce apoptosis of cultured and primary CD34+ human AML blast progenitor cells (BPC) expressing FLT3-ITD. Concomitantly, as compared with each agent alone, cotreatment with JQ1 and the FLT3-TKI caused greater attenuation of c-MYC, BCL2, and CDK4/6. Simultaneously, cotreatment with JQ1 and the FLT3-TKI increased the levels of p21, BIM, and cleaved PARP, as well as mediated marked attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels in AML BPCs. Conversely, cotreatment with JQ1 and FLT3-TKI was significantly less active against CD34+ normal bone marrow progenitor cells. Knockdown of BRD4 by short hairpin RNA also sensitized AML cells to FLT3-TKI. JQ1 treatment induced apoptosis of mouse Ba/F3 cells ectopically expressing FLT3-ITD with or without FLT3-TKI–resistant mutations F691L and D835V. Compared with the parental human AML FLT3-ITD–expressing MOLM13, MOLM13-TKIR cells resistant to AC220 were markedly more sensitive to JQ1-induced apoptosis. Furthermore, cotreatment with JQ1 and the pan-histone deacetylase inhibitor (HDI) panobinostat synergistically induced apoptosis of FLT3-TKI–resistant MOLM13-TKIR and MV4-11-TKIR cells. Collectively, these findings support the rationale for determining the in vivo activity of combined therapy with BA and FLT3-TKI against human AML cells expressing FLT3-ITD or with BA and HDI against AML cells resistant to FLT3-TKI. Mol Cancer Ther; 13(10); 2315–27. ©2014 AACR.

Synergistic activity of BET protein antagonist-based combinations in mantle cell lymphoma cells sensitive or resistant to ibrutinib

Mantle cell lymphoma (MCL) cells exhibit increased B-cell receptor and nuclear factor (NF)-κB activities. The bromodomain and extra-terminal (BET) protein bromodomain 4 is essential for the transcriptional activity of NF-κB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and cyclin-dependent kinase (CDK)4/6, inhibits the nuclear RelA levels and the expression of NF-κB target genes, including Bruton tyrosine kinase (BTK) in MCL cells. Although lowering the levels of the antiapoptotic B-cell lymphoma (BCL)2 family proteins, BA treatment induces the proapoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Cotreatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared with each agent alone, cotreatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells, which overexpress CDK6, BCL2, Bcl-xL, XIAP, and AKT, but lack ibrutinib resistance-conferring BTK mutation. Cotreatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT-199 (BCL2 antagonist) synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL.

Pre-clinical efficacy of combined therapy with novel β-catenin antagonist BC2059 and histone deacetylase inhibitor against AML cells

The canonical wingless-type MMTV integration site (WNT)-β-catenin pathway is essential for self-renewal, growth and survival of acute myeloid leukemia (AML) stem/blast progenitor cells (BPCs). Deregulated WNT signaling inhibits degradation of β-catenin, causing increased nuclear translocation and co-factor activity of β-catenin with the transcriptional regulator T-cell factor (TCF) 4/lymphoid enhancer factor 1 in AML BPCs. Here, we determined the pre-clinical anti-AML activity of the anthraquinone oxime-analog BC2059 (BC), known to attenuate β-catenin levels. BC treatment disrupted the binding of β-catenin with the scaffold protein transducin β-like 1 and proteasomal degradation and decline in the nuclear levels of β-catenin. This was associated with reduced transcriptional activity of TCF4 and expression of its target genes, cyclin D1, c-MYC and survivin. BC treatment dose-dependently induced apoptosis of cultured and primary AML BPCs. Treatment with BC also significantly improved the median survival of immune-depleted mice engrafted with either cultured or primary AML BPCs, exhibiting nuclear expression of β-catenin. Co-treatment with the pan-histone deacetylase inhibitor panobinostat and BC synergistically induced apoptosis of cultured and primary AML BPCs, including those expressing FLT3-ITD, as well as further significantly improved the survival of immune-depleted mice engrafted with primary AML BPCs. These findings underscore the promising pre-clinical activity and warrant further testing of BC against human AML, especially those expressing FLT3-ITD.

HEXIM1 induction is mechanistically involved in mediating anti-AML activity of BET protein bromodomain antagonist.

HEXIM1 induction is mechanistically involved in mediating anti-AML activity of BET protein bromodomain antagonist

Abstract 3686: HEXIM1 induction exerts a mechanistic role and is a biomarker of lethal activity of BRD4 antagonist against human AML cells

The BET protein BRD4 binds to acetylated lysines on the histone proteins, recruits pTEFb kinase, a complex of cyclin T and CDK9, to gene promoters to phosphorylate serine 2 of the C-terminus of RNA pol (RNAP) II. This promotes the pause release of RNAP II, with the elongation and expression of the mRNA transcripts of several oncogenes as well as their target genes, which are essential for the growth and survival of AML cells. We have recently determined that the BET protein antagonist JQ1, which disrupts the binding of BRD4 to acetylated lysines, potently induces apoptosis in cultured (OCI-AML3 and MOLM13) and patient-derived primary AML blast progenitor cells. JQ1 treatment also improved the survival of the immune-depleted mice engrafted with MOLM13 or primary AML cells. The lethal activity of JQ1 is associated with marked induction of hexamethylene bisacetamide (HMBA)-inducible protein 1 (HEXIM1) and p21, while MYC, BCL2 and CDK4/6 expressions are concomitantly downregulated in the cultured and primary AML cells. HEXIM1 binds and sequesters pTEFb, thereby inhibiting its kinase activity for RNAP II. To determine the role of HEXIM1 induction on the lethal effects of JQ1, we achieved a stable lentivirus mediated knockdown (KD) of the mRNA and protein expressions of HEXIM1 by approximately 90% in OCI-AML3 and MOLM13 cells. There was no significant difference in the protein expressions of BRD4, MYC and BCL2, or of cyclin T and CDK9 levels in the nuclear fraction in the AML cells with HEXIM1 knockdown (HKD) versus the AML cells expressing non-targeted (NT) shRNA. However, in the HKD cells, higher levels of the complex of cyclin T with CDK9 as pTEFb were detected in the immunoprecipitates of cyclin T. As compared to the NT AML, JQ1 (250 to 2000 nM)-induced HEXIM1 and apoptosis was reduced in HKD AML cells. This was associated with abrogation of JQ1-mediated HEXIM1 and p21 induction in the HKD AML cells. Similarly, HMBA (5 mM)-induced HEXIM1 and apoptosis was also inhibited in the HKD versus NT AML cells. Co-treatment with JQ1 and pan-HDAC inhibitor panobinostat (PS) synergistically induced apoptosis of cultured (MOLM13 and OCI-AML3) and primary AML blast progenitor cells, as well as conferred superior in vivo survival on the mice engrafted with cultured and primary AML blast progenitor cells. Importantly, co-treatment with JQ1 and PS also synergistically induced apoptosis of the cultured HKD AML cells. These findings indicate that HEXIM1 induction is a biomarker and plays a mechanistic role in the lethal activity of BRD4 antagonist against human AML cells. Citation Format: Santhana G. T. Devaraj, Warren Fiskus, Sunil Sharma, Jun Qi, Bhavin Shah, Leasha J. Schaub, Melissa Rodriguez, Ka Liu, Swaminathan P. Iyer, James E. Bradner, Kapil N. Bhalla. HEXIM1 induction exerts a mechanistic role and is a biomarker of lethal activity of BRD4 antagonist against human AML cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3686. doi:10.1158/1538-7445.AM2014-3686

Abstract 1721: BRD4 antagonist-based therapy exerts lethal activity against FLT3 mutation expressing AML cells resistant to FLT3 tyrosine kinase inhibitors

The BET (bromodomain and extra terminal) protein antagonists, e.g., JQ1, disrupt the binding of the bromodomains of BRD4 to acetylated lysines, displacing the BET proteins, along with the associated transcript initiation and elongation factors, from the chromatin. JQ1 treatment also induces hexamethylene bisacetamide (HMBA)-inducible protein 1 (HEXIM1), which binds and sequesters pTEFb, thereby inhibiting its kinase activity for RNAP II. This inhibits the pause release of RNAP II, thereby attenuating the elongation and expression of the mRNA of oncogenes, e.g., c-MYC and BCL2, and their target genes, leading to growth inhibition and apoptosis of AML cells. Although the second generation FLT3 tyrosine kinase inhibitors (TKI), such as AC220 induce remissions in the aggressive sub-type of AML expressing FLT3 mutations, resistance-causing gatekeeper (F691I/L), activation loop (AL) (D835V/Y/F) or compound FLT3-ITD/F691I mutations are known to impair the in vitro and in vivo activity of the FLT3-TKIs. We have recently determined that JQ1, but not its inactive enantiomer R-JQ1 potently induced apoptosis of not only Ba/F3/FLT3-ITD but also Ba/F3/FLT3-ITD expressing the highly FLT3 TKI-resistant mutations F691L and D835V (IC 50 values for JQ1 were 697, 1588 and 909 nM, in the three cell lines, respectively). JQ1 dose-dependently induced apoptosis of human cultured AML MOLM13 and MV4-11 cells, as well as of primary AML cells, all expressing FLT3-ITD. Concomitantly, JQ1 treatment attenuated c-MYC, BCL2 and CDK6, while inducing p21, p27, BIM and cleaved PARP levels. Following engraftment of NOD/SCID mice with MOLM13 xenografts, treatment with JQ1 (50 mg/kg, daily x 5 days per week x 3 weeks), versus treatment with vehicle control, significantly improved survival of the mice (p 50-fold resistance to AC220. As compared to the parental MOLM13, the TKI-resistant cells express higher levels of BRD4, c-MYC and class I HDACs. Importantly, as compared to the parental MOLM13, the MOLM13/TKIR cells were significantly more sensitive to JQ1-induced apoptosis (p Citation Format: Melissa Rodriguez, Warren Fiskus, Sunil Sharma, Jun Qi, Leasha J. Schaub, Bhavin Shah, Santhana G.T. Devaraj, Ka Liu, Swaminathan Iyer, James E. Bradner, Kapil N. Bhalla. BRD4 antagonist-based therapy exerts lethal activity against FLT3 mutation expressing AML cells resistant to FLT3 tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1721. doi:10.1158/1538-7445.AM2014-1721

Abstract 3243: Development of specific DNA aptamers against programmed cell death-1 (Anti-PD1-Apt) for diagnosis and treatment of cancers

Cancer cells have an extraordinary ability to escape immune response by modulating proteins that regulate immune checkpoints and their cognate ligands. Programmed cell death protein (PD-1), a cell surface receptor expressed on T cells is one such immune checkpoint receptor when bound to its ligands- PDL1 or PDL2 transmits an inhibitory signal. Such modulation often leads to inhibition of T-cell activation and subsequent escape of tumors from immune surveillance. Recently, several FDA approved therapeutic antibodies have been successfully developed that target PD-1/PD-L1 axis and allow the immune system to enhance its anti-cancer effects. Aptamers are synthetic small molecule ligands composed of short, single-stranded oligonucleotides ranging from 30 to 60 bases in length. Based on their highly specific 3-dimensional conformation, aptamers, analogous to the antibodies can recognize and bind to their targets with high affinity. Also, the nucleic acid component has several advantages over the protein counterparts- such as ease of production under less stringent conditions, long shelf life and low cost. Here, we report the development of several PD-1 specific aptamers by systematic evolution of ligands by exponential enrichment (SELEX) technology against endogenous immunoprecipitated PD-1 protein using DNA library with a complexity of 1014. Following several rounds of SELEX, the selected aptamers sequenced by high throughput Next-Generation Sequencing (NGS) were found to have highly conserved regions. Six PD-1 specific aptamers (Anti-PD1-Apt) were then assessed for target validation using leukemic cell lysates (cell lines and primary patient samples) and were found to bind to the PD-1 in its native state. The selected Anti-PD1-Apt were able to specifically pull down the PD-1 protein from the lysates mimicking PD-1 antibody. The specific interaction of the Anti-PD1-Apt was also demonstrated by flow cytometry and fluorescent microscopy. As expected, Anti-PD1-Apt was able to bind to PD-1 with Kd of ~ 500 picomolar affinity as assessed by Bio-Layer Interferometry. Furthermore, we also characterized and confirmed Anti-PD1-Apt biological activity using an PD-1/PD-L1 cell-based assay using PD-1/NFAT reporter-Jurkat cells. We have observed several fold induction of NFAT luciferase reporter activity (Relative Luciferase Units) in PD-1/NFAT reporter-Jurkat cells co-cultured with HEK293 cells overexpressing PD-L1 and TCR activator in the presence of Anti-PD1-Apt compared to control. Our preliminary data also demonstrate robust Anti-PD1 blockade in Mixed Lymphocyte Reaction (MLR) along with induction of Th1 cytokines Interferon-gamma and IL-2 from different donor sets of PBMCs. We will present additional - in vivo antitumor response data at the upcoming AACR Annual Meeting at Washington DC, 2017. Citation Format: Santhana Gowri Thangavelu Devaraj, Lokesh Ganesh Lakshmana Rao, Youli Zu, Jenny C. Chang, Swaminathan P. Iyer. Development of specific DNA aptamers against programmed cell death-1 (Anti-PD1-Apt) for diagnosis and treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3243. doi:10.1158/1538-7445.AM2017-3243

Abstract A124: Co-treatment with BRD4 antagonist and histone deacetylase inhibitor is synergistically lethal against Mantle Cell Lymphoma (MCL) cells.

The BET (bromodomain and extraterminal) protein family members including BRD4 bind to acetylated lysines on the histone proteins, help assemble transcriptional regulators at the target gene promoters and enhancers, and regulate the expression of important oncogenes, e.g., Myc and BCL-2. Here we determined the effects of the BET protein inhibitor JQ1 and/or histone deacetylase (HDAC) inhibitor panobinostat (PS) on cultured (JeKo1 and MO2058) and primary human MCL cells harvested from the excised MCL involved lymph nodes. Treatment with JQ1 (100 to 2000 nM), but not its inactive enantiomer (R-JQ1), dose-dependently increased the % of cells in the G1 phase while reducing the % of S phase cells, while concomitantly inducing apoptosis in the cultured (MO2058 > JeKo1) MCL cells. Treatment with JQ1 was also dose-dependently lethal against primary MCL cells. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of MYC, BCL2 and CDK6 promoter in JeKo1 and MO2058 cells. Total RNA from the untreated and JQ1-treated cells was used for the quantitative PCR analysis, which showed depletion of the mRNA of c-MYC, BCL2 and CDK6 genes in JQ1-treated cells. While it had no effect on acetylated histone H3 and BRD4, JQ1 treatment dose-dependently depleted the protein levels of MYC, BCL2, CDK6 and pSer2 RNA POL II, but induced the levels of p21, p27 and cleaved PARP in MCL cells. As compared to each agent alone, co-treatment with JQ1 (but not its inactive enantiomer, R-JQ1) and panobinostat (PS) synergistically induced apoptosis of the cultured and primary MCL cells (combination indices 10 fold resistant), which were isolated following selection under a continuous exposure to increasing levels of carfilzomib. Following the tail vein infusion and engraftment of JeKo1 cells (5 million cells/mouse) in the bone marrow and spleen of NOD/SCID mice, co-treatment with JQ1 (50 mg/kg/day, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, administered IP) and PS (5 mg/kg, IP) versus treatment with vehicle control, or JQ1 or PS alone, resulted in significant in vivo attenuation of c-MYC, BCL-2 and cyclin D1 levels in the harvested MCL cells from the mice (p < 0.01). Collectively, these pre-clinical findings demonstrate that the combined treatment with BRD4 antagonist and pan-HDAC inhibitor is a synergistically effective epigenetic therapy targeted against human MCL cells, regardless of their sensitivity to proteasome inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A124. Citation Format: Bhavin Shah, Laxmi Jakkula, Warren Fiskus, Sunil Sharma, Jun Qi, John A. Valenta, Leasha J. Schaub, Melissa Rodriguez, Santhana G.T. Devaraj, James E. Bradner, Kapil N. Bhalla. Co-treatment with BRD4 antagonist and histone deacetylase inhibitor is synergistically lethal against Mantle Cell Lymphoma (MCL) cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A124.