Discovery platform for inhibitors of IgH gene enhancer activity

Abstract

Immunoglobulin heavy chain (IgH) translocations are common and early oncogenic events in B cell and plasma cell malignancies including B cell non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). IgH translocations bring oncogenes into close proximity with potent enhancer elements within the IgH locus, leading to oncogene up-regulation. As IgH enhancer activity is tightly controlled by B cell lineage-specific signaling and transcriptional networks, we hypothesized that IgH enhancers are potentially druggable targets/elements. To test this, we developed a molecular imaging-based high-throughput screening platform for discovering inhibitors of IgH enhancer-driven transcriptional activity. As proof of concept, we identified a low micromolar potency molecule (compound 30666) that inhibited immunoglobulin production by MM cells and blocked expression of an array of IgH translocation-induced oncogenes (CCND1, FGFR3/MMSET, and MYC) in MM and NHL cell lines. Prolonged exposure to 30666 significantly reduced the viability of IgH translocation-positive NHL and MM cells, but was less effective against cells lacking IgH translocations. Compound 30666 exhibited suitable pharmacological properties, including metabolic stability in liver microsomes and oral bioavailability in mice, and demonstrated preclinical anti-MM activity in a plasmacytoma mouse model. Our work suggests that IgH enhancers are attractive and potentially druggable targets for IgH translocation driven malignancies.

Keywords: IgH Gene Translocation; Multiple myeloma; cancer biology; cytogenetic; gene enhancer; immunoglobulin; non-hodgkins lymphoma; oncogene.

Figure 1.

Compound 30666 inhibits IgH gene enhancer transcriptional activity. (A) The indicated cell lines were transfected with a control firefly luciferase reporter (pGL3) or a reporter driven by a variable IgH promoter and Eμ intronic IgH enhancer sequence (IGHE’-Luc). Reporter activity is shown as folds above control reporter. A representative bioluminescence image is shown on the right.(B) IgH enhancer activity was measured following treatment with agents that are approved for the treatment of MM (bortezomib, Btz, 5 nM; dexamethasone, Dexa, 10 μM; lenalidomide, Lenalid, 100 μM) or the multi-kinase inhibitor sorafenib (2 μM). A control reporter is shown as an internal standard to account for any non-specific drug activity. Compound 30666 was included as a positive control.(C) BJAB cells expressing IgH enhancer driven reporter (IGHE’-Luc) or control reporter were treated with a dose range of compound 30666 for 16 hours. Reporter activity is shown as a percentage of control (DMSO) treated cells. The chemical structure of compound 30666 (7-[[(4-methyl-2-pyridinyl)amino](2-pyridinyl)methyl]-8-quinolinol, C₂₁H₁₈N₄O) is shown in the inset.(D) BJAB cells expressing IGHE’-Luc (left panel) or control reporter (right panel) were treated with a dose range of 30666 derivatives for 16 hours. Reporter activity is shown as a percentage of control (DMSO) treated cells. Chemical structures for the 30666 derivatives are shown with modifications highlighted in red.

Figure 2.

Lead compound 30666 represses the expression of IgH translocation induced oncogenes. (A) U266 MM cells harboring the t(11;14) IgH translocation, which results in the overexpression of the CCND1 oncogene, were treated with a dose range (0, 0.32, 0.63, 1.25, 2.5, 5.0 μM) of 30666. Western blots are shown. The MM standard of care agent, Btz (5 nM), was included for comparison.(B) t(11;14) positive Granta519 MCL cells were treated as in (A). Western blots are shown.(C) [Left panel] KMS18 MM cells, which harbor the t(4;14) reciprocal translocation resulting in the up-regulation of FGFR3 and MMSET oncogenes, were treated as in (A) and (B). Western blots are shown.[Right panel] 595sp mouse MM cells, which overexpress MYC and BCL2L1 due to gene rearrangements with the mouse IGH and IGL genes, respectively, were treated with a dose range of 30666 as in (A). Western blots are shown.(D) Ramos cells, which overexpress MYC due to the t(8;14) IgH translocation, were treated with 30666 (3 μM) for 24 hours. MYC mRNA was quantified relative to GAPDH by qPCR (*P < 0.05). U266 MM cells were also treated with 30666 (3μM) for 24 hours and CCND1 mRNA levels measured by qPCR (*P < 0.05).(E) [Top panel] K562 chronic myelogenous leukemia (CML) cells, which express the BCR-Abl oncogene due to the presence of the Philadelphia chromosome, were treated with increasing concentrations of 30666 for 24 hours. Western blots are shown.[Bottom panel] Karpas 299 cells, a t(2;5)-positive anaplastic T cell lymphoma cell line that expresses a fusion NPM-ALK gene, were treated with increasing concentrations of 30666 for 24 hours. Western blots are shown.(F) IgG secreting SA13 human hybridoma cells were treated with a dose range of 3066 (0, 1.25, 2.5, 5.0, 10 μM) for 24 hours. Western blots are shown at left (HC: Ig heavy chain; LC: Ig light chain).(G) IgE secreting U266 MM cells were treated as in (E). Western blots (left) and viable cell counts (right) are shown.

Figure 3.

Compound 30666 exhibits potent and selective activity against hematological cancer cells harboring IgH gene translocations. (A) The indicated cell lines were treated with a dose range of 30666 for 48 hours. Cell viability data is shown. Cells marked by red regression lines and type are positive for IgH translocations [IgH Tx(+)] whereas cell lines marked in black are negative [IgH Tx(-)].(B) EC50 values were extrapolated from dose curves in (A). A student’s t-test was used to measure statistical significance.(C) The indicated cell lines were treated with DMSO (control) or 30666 (3 μM) for 48 hours. Cell death was measured by propidium iodide staining and flow cytometric analysis of DNA content. Data is represented as the percentage of dead cells (i.e., those with sub-G1 DNA content) per population.(D) Parental MM.1S cells or Btz resistant MM.1S cells (MM.1S BzR) were treated with a dose range of 30666 (top panel) or Btz (bottom panel). Cell viability data are shown.(E) CD138+ primary MM patient plasma cells were isolated from bone marrow aspirates and cultured in the presence of 30666 or dexamethasone. Cell viability data are shown for a 48 hour time point.

Figure 4.

Pharmacokinetics and in vivo anti-MM efficacy of compound 30666. (A) C57BL/6 mice were given a single dose of 5 mg/kg 30666 via oral gavage (PO, black circles) or intraperitoneal (IP, red squares) injection. Blood was drawn at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after dosing, and plasma concentrations of 30666 were quantified by LC-MS/MS. The average (mean ± S.E.) plasma concentration is shown plotted against time for each route of administration (N = 3 mice per time point).(B) The metabolic stability of 30666 was determined by incubating the compound in the presence of human liver microsomes. The disappearance of 30666 or control compound (terfenadine) was measured by LC-MS/MS at 0, 5, 15, 30, and 60-minute intervals.(C) Plasmacytomas were induced in SCID Hairless Outbred mice by subcutaneous injection of RPMI-8226 human MM cells. Once tumors reached a plapable size of 150 mm³, treatments with daily 30666 (5 mg/kg, i.p.) or vehicle (PBS + 5% Cremophor) were initiated. Mice were treated for 14 days. Tumor volume data are shown at the study endpoint (28 days post initiation of drug treatment). A student’s t-test statistical analysis was used (N = 10).(D) Body weight analysis from animals treated in (C) is shown. Animals were weighed prior to treatment with 30666 (Pre-Rx) and on day 15 after 14 days of 30666 treatment (Post-Rx). There was no significant difference in body weight between groups either before or after drug treatment (N = 5).