N-quinoline-benzenesulfonamide derivatives exert potent anti-lymphoma effect by targeting NF-κB.

We previously identified the N-quinoline-benzenesulfonamide (NQBS) scaffold as a potent inhibitor of nuclear factor-κB (NF-κB) translocation. Now, we report the structure-activity relationship of compounds with the NQBS scaffold in models of diffuse large B-cell lymphoma (DLBCL). We identified CU-O42, CU-O47, and CU-O75 as NQBS analogs with the most potent cytotoxic activity in DLBCL lines. Their anti-lymphoma effect was mediated by NF-κB sequestration to the cytoplasm of DLBCL cells. Internal Coordinates Mechanics analysis suggested direct binding between CU-O75 and IκBα/p50/p65 which leads to the stabilization of the NF-κB trimer. A whole cellular thermal shift assay confirmed direct binding of the NQBS to IκBα, an inhibitory component of the IκBα/p50/p65 trimer. Lymphoma cell line sequencing revealed CU-O75 induced downregulation of NF-κB-dependent genes and DeMAND analysis identified IκBα as one of the top protein targets for CU-O75. CU-O42 was potent in inhibiting tumor growth in two mouse models of aggressive lymphomas.

Generation of pralatrexate resistant T-cell lymphoma lines reveals two patterns of acquired drug resistance that is overcome with epigenetic modifiers.

While pralatrexate (PDX) has been successfully developed for the treatment of T-cell lymphoma, the mechanistic basis for its T-cell selectivity and acquired resistance remains elusive. In an effort to potentially identify synergistic combinations that might circumnavigate or delay acquired PDX resistance, we generated resistant cells lines over a broad concentration range. PDX-resistant cell lines H9-12 and H9-200 were developed, each exhibiting an IC50 of 35 and over 1000 nM, respectively. These lines were established in vitro from parental H9 cells. Expression analysis of the proteins known to be important determinants of antifolate pharmacology revealed increase expression of dihydrofolate reductase (DHFR) due to gene amplification, and reduced folate carrier1 downregulation, as the putative mechanisms of resistance in H9-12 and H9-200 cells. Cross resistance was only seen with methotrexate but not with romidepsin, azacitidine (AZA), decitabine, gemcitabine, doxorubicin, or bortezomib. Resistance to PDX was reversed by pretreatment with hypomethylating agents in a concentration-dependent fashion. Comparison of gene expression profiles of parental and resistant cell lines confirmed markedly different patterns of gene expression, and identified the dual specificity phosphatase four (DUSP4) as one of the molecular target of PDX activity. Reduced STAT5 phosphorylation following exposure to PDX was observed in the H9 but not in the H9-12 and H9-200 cells. These data suggest that combination with hypomethylating agents could be potent, and that DUSP4 and STAT5 could represent putative biomarkers of PDX activity.

The anti-tumor activity of pralatrexate (PDX) correlates with the expression of RFC and DHFR mRNA in preclinical models of multiple myeloma.

Multiple myeloma (MM) is the second most common hematologic malignancy. While major advances have been made in the disease, it is still incurable. Although antifolate-based drugs are not commonly used to treat myeloma, new generation analogs with distinct patterns of preclinical and clinical activity may offer an opportunity to identify new classes of potentially active drugs. Pralatrexate (PDX), which was approved for the treatment of relapsed or refractory peripheral T-cell lymphoma in 2009, may be one such drug. Pralatrexate exhibits a potency and pattern of activity distinct from its predecessors like methotrexate (MTX). We sought to understand the activity and mechanisms of resistance of multiple myeloma to these drugs, which could also offer potential strategies for selective use of the drug. We demonstrate that PDX and MTX both induce a significant decrease in cell viability in the low nanomolar range, with PDX exhibiting a more potent effect. We identified a series of myeloma cell lines exhibiting markedly different patterns of sensitivity to the drugs, with some lines frankly resistant, and others exquisitely sensitive. These differences were largely attributed to the basal RFC (Reduced Folate Carrier) mRNA expression levels. RFC mRNA expression correlated directly with rates of drug uptake, with the most sensitive lines exhibiting the most significant intracellular accumulation of pralatrexate. This mechanism explains the widely varying patterns of sensitivity and resistance to pralatrexate in multiple myeloma cell lines. These findings could have implications for this class of drugs and their role in the treatment of multiple myeloma.

Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kδ and CK1ε in hematological malignancies.

Phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells. TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc-dependent transcription. The synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kδ inhibitors, inhibited casein kinase-1 ε (CK1ε). Targeting CK1ε using a selective chemical inhibitor or short hairpin RNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. These results suggest that TGR-1202 is a dual PI3Kδ/CK1ε inhibitor, which may in part explain the clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1ε should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc.

Aurora A Kinase Inhibition Selectively Synergizes with Histone Deacetylase Inhibitor through Cytokinesis Failure in T-cell Lymphoma.

PURPOSE: Aurora A kinase (AAK) is expressed exclusively during mitosis, and plays a critical role in centrosome duplication and spindle formation. Alisertib is a highly selective AAK inhibitor that has demonstrated marked clinical activity of alisertib across a spectrum of lymphomas, though particularly in patients with T-cell lymphoma (TCL). We sought to compare and contrast the activity of alisertib in preclinical models of B-cell lymphoma (BCL) and TCL, and identify combinations worthy of clinical study. High-throughput screening of pralatrexate, the proteasome inhibitor (ixazomib), and the histone deacetylase (HDAC) inhibitor (romidepsin) revealed that only romidepsin synergized with alisertib, and only in models of TCL. We discovered that the mechanism of synergy between AAK inhibitors and HDAC inhibitors appears to be mediated through cytokinesis failure. EXPERIMENTAL DESIGN: A high-throughput screening approach was used to identify drugs that were potentially synergistic in combination with alisertib. Live-cell imaging was used to explore the mechanistic basis for the drug: drug interaction between alisertib and romidepsin. An in vivo xenograft TCL model was used to confirm in vitro results. RESULTS: In vitro, alisertib exhibited concentration-dependent cytotoxicity in BCL and TCL cell lines. Alisertib was synergistic with romidepsin in a T-cell-specific fashion that was confirmed in vivo. Live-cell imaging demonstrated that the combination treatment resulted in profound cytokinesis failure. CONCLUSIONS: These data strongly suggest that the combination of alisertib and romidepsin is highly synergistic in TCL through modulation of cytokinesis and merits clinical development.

Pralatrexate pharmacology and clinical development.

Folates are well known to be essential for many cellular processes, including cellular proliferation. As a consequence, antifolates, the fraudulent mimics of folic acid, have been shown to be potent therapeutic agents in many cancers. Over the past several decades, efforts to improve on this class of drugs have met with little success. Recently, one analog specifically designed to have high affinity for the reduced folate carrier, which efficiently internalizes natural folates and antifolates, has been shown to be very active in T-cell lymphoma. Pralatrexate, approved by the U.S. Food and Drug Administration in 2009, is highly active across many lymphoid malignancies, including chemotherapy-resistant T-cell lymphoma. Emerging combination studies have now shown that pralatrexate is highly synergistic with gemcitabine, histone deacetylase inhibitors like romidepsin and bortezomib. These insights are leading to a number of novel phase I and II combination studies which could challenge existing regimens like CHOP, and improve the outcome of patients with T-cell lymphoma.

Cellular immune responses against CT7 (MAGE-C1) and humoral responses against other cancer-testis antigens in multiple myeloma patients.

The type I melanoma antigen gene (MAGE) proteins CT7 (MAGE-C1) and MAGE-A3 are commonly expressed in multiple myeloma (MM), and their expression correlates with increased plasma cell proliferation and poor clinical outcome. They belong to the cancer-testis antigen (CTAg) group of tumor-associated proteins, some of which elicit spontaneous immune responses in cancer patients. CT7 and MAGE-A3 are promising antigenic targets for therapeutic tumor vaccines in myeloma; therefore, it is critical to determine if they are immunogenic in MM patients. We analyzed cellular and humoral immune responses against CTAgs in patients with plasma cell dyscrasias: MM, monoclonal gammopathy of undetermined significance (MGUS), and Waldenström's macroglobulinemia (WM). Bone marrow lymphocytes from two of four untreated MM patients exhibited CT7-specific cellular immune responses as measured by an autologous cellular immunity assay, the first such immune response to CT7 to be reported in cancer patients. Sera from 24 patients were screened by ELISA for humoral immune responses to CTAgs. Two patients with MM demonstrated positive titers, one for MAGE-A1 and the other for SSX1. These data demonstrate that CTAgs, particularly CT7, are immunogenic in MM patients and merit further exploration as targets of immunological therapy in MM.